Engineered cells and methods of use

ABSTRACT

Provided herein are engineered cells, comprising: a chemical or biological moiety covalently bound to a cell surface glycan, wherein the chemical or biological moiety is selected from the group consisting of small molecule, polynucleotide, polypeptide, and antibody. Also provided are compositions comprising these engineered cells and methods of making and using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

The subject patent application is a divisional of U.S. Pat. ApplicationSerial No. 16/482,497 (filed Jul. 31, 2019; now pending), which is a§371 U.S. national phase filing of PCT International Patent ApplicationNo. PCT/US2018/016503 (filed Feb. 1, 2018; now expired), which claimsthe benefit of priority to U.S. Provisional Pat. Application Ser. No.62/453,922 (filed Feb. 2, 2017; now expired) and U.S. Provisional Pat.Application Ser. No. 62/578,721 (filed on Oct. 30, 2017; now expired).The full disclosures of the priority applications are incorporatedherein by reference in their entirety and for all purposes.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with government support under GM113046 andGM093282 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to engineered and modified cells,specifically in the field of medicine.

BACKGROUND OF THE DISCLOSURE

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Cancer is a leading cause of death in the United States and worldwide.Recently, immunotherapy has emerged as a fourth weapon besides surgery,radiation, and chemotherapy deployed for the treatment of cancer. Cancerimmunotherapy aims to harness the body’s own immune system to fightcancer. While good results have been achieved in adoptive cell therapy(ACT) and checkpoint blockade therapy, the cancer fighting properties ofthe T-cells infused in the patients remain to be improved, includingtheir migratory potentials and capabilities to counteract theimmune-suppressive signals in the tumor microenvironment. Natural killer(NK) cells play a crucial role in innate immunity against malignantcells, yet also being developed as an effective cancer killer in cancerimmunotherapy. However, one challenge of NK cell based therapy is thedifficulty of obtaining sufficient numbers of active NK cells from apatient’s blood. NK-92, a highly cytotoxic natural killer (NK) cell lineestablished from patients with clonal NK-cell lymphoma, can be employedto generate larger numbers of cytotoxic NK cells in GMP-grade. Specifictargeting is a gold standard for a good cancer therapy. However, NK-92cells do not express Fc receptors for ADCC effects to target specificcells, which limit its wide applications.

Likewise, dendritic cells (DCs) have long been considered as the centralplayers in cancer immunotherapy. The migratory ability of DCs hasprofound impact on the outcome of the DC-based immunotherapy. However,it was shown that only a small portion (1-2%) of total administered DCsreached secondary lymphatic organs to activate T cells, limiting thepractical application of this vaccination approach.

Thus there remains a need in the art for new compositions and methodsthat can effectively label antigen specific antibody onto a cell surfacewhich in turn directs these cells to kill specific antigen expression indiseased cells and effectively treat diseases.

SUMMARY OF THE DISCLOSURE

Various embodiments disclosed herein include an engineered cell,comprising a chemical or biological moiety covalently bound to a cellsurface glycan present on the surface of the cell, wherein the chemicalor biological moiety is selected from the group consisting of smallmolecule, polynucleotide, polypeptide, and antibody. In one embodiment,the engineered cell is an immune cell. In one embodiment, the engineeredcell is a T-cell or a natural killer (NK) cell. In one embodiment, the Tcell is a CD8+ or a CD4+ T cell. In one embodiment, the cell is aDendritic Cell (DC). In one embodiment, the small molecule is a smalldrug molecule or a pharmaceutically acceptable salt or a co-crystalthereof. In one embodiment, the antibody is a single chain variablefragment (scFv), fragment antigen binding (Fab) fragment, or a fulllength antibody. In one embodiment, the antibody is an immunoglobulin G(IgG) antibody. In one embodiment, the IgG is a full length IgG. In oneembodiment, the chemical or biological moiety attached to the engineeredcell is a biological marker and/or probe. In one embodiment, thechemical or biological moiety is a biotin probe, a fluorescent probe, abiorthogonal reaction handle, and/or a dye labeled single strand DNA. Inone embodiment, the dye is FAM. In one embodiment, the fluorescent probeis Cy3. In one embodiment, the biorthogonal reaction handle istetrazine. In one embodiment, the fucose derivative is a GDP-fucose. Inone embodiment, the engineered cell is a chimeric antigen receptor(CAR)-T cell. In one embodiment, the CAR-T cell comprises a geneticallymodified T-cell with the cell surface GlcNAc covalently bound to aGDP-Fucose bearing a new motif. In one embodiment, the CAR comprisesthree domains: scFv, Fab, and/or mature ligands that engage theircognate receptor. In one embodiment, the cell surface glycan isN-acetylglucosamine (GlcNAc). In one embodiment, the chemical orbiological moiety is covalently bound to the GlcNAc via a fucosederivative. In one embodiment, the cell surface glycan is Sialic acid(NeuAc).

Various embodiments disclosed herein also include a compositioncomprising: an antibody-cell conjugate, wherein one or more antibodiesare covalently bound to one or more glycan moiety on the surface of acell. In one embodiment, the cell is immune cell. In one embodiment, thecell is a primary human T cell, a natural killer (NK) cell, a CD4+ cell,and/or primary CD8+ OT-1 T cells. In one embodiment, the NK cell isNK-92MI cell. In one embodiment, the cell is a Dendritic Cell (DC). Inone embodiment, the antibody is Trastuzumab. In one embodiment, the cellis NK-92MI and at least one antibody is Trastuzumab. In one embodiment,more than one type of antibody is conjugated on the surface of the cell.In one embodiment, the glycan moiety is N-acetylglucosamine (GlcNAc). Inone embodiment, the antibody is covalently bound to the GlcNAc via afucose derivative. In one embodiment, the glycan moiety is Sialic acid(NeuAc). In one embodiment, the composition is a pharmaceuticalcomposition. In one embodiment, the pharmaceutical composition furthercomprises a pharmaceutically acceptable carrier or excipient. In oneembodiment, the pharmaceutical composition is for the treatment of adisease in a subject. In one embodiment, the antibody-cell conjugateenables firm binding on inflammation sites (anti-E-selectin), targetspecific cancer cells (anti-HER2), and/or block the immune checkpoint(anti-PD-L1).

Various embodiments disclosed herein further include a method oftreating a disease in a subject comprising: providing a pharmaceuticalcomposition comprising an engineered cell and a pharmaceuticallyacceptable carrier; and treating the disease by administering atherapeutically effective dosage of the pharmaceutical composition tothe subject, wherein the engineered cell comprises a chemical orbiological moiety covalently bound to a glycan moiety present on itssurface, and wherein the chemical or biological moiety is selected fromthe group consisting of small molecule, polynucleotide, polypeptide, andantibody. In one embodiment, the antibody is an immunoglobulin G (IgG)antibody. In one embodiment, the antibody is an anti-PD-L1 antibody. Inone embodiment, the antibody is an anti-E-selectin antibody. In oneembodiment, the antibody is an anti-HER2 antibody. In one embodiment,the antibody is Trastuzumab. In one embodiment, more than one chemicalor biological moiety is covalently bound to the surface of the cell totarget more than one disease at the same time. In one embodiment, thecell is a dendritic cell. In one embodiment, the cell is an immune cell.In one embodiment, the cell is a primary human T cell, a natural killer(NK) cell, a CD4+ cell, and/or primary CD8+ OT-1 T cells. In oneembodiment, the NK cell is NK-92MI cell. In one embodiment, the glycanmoiety is N-acetylglucosamine (GlcNAc). In one embodiment, the chemicalor biological moiety is covalently bound to the GlcNAc via a fucosederivative. In one embodiment, the fucose derivative comprisesfucose-alkyne. In one embodiment, the glycan moiety is Sialic acid(NeuAc). In one embodiment, the disease is cancer. In one embodiment,treating the disease comprises reducing the size of a cancerous tumor inthe subject. In one embodiment, the cancer is a breast cancer.

Embodiments of the present disclosure also include a method of treating,decreasing, inhibiting, or reducing cancer in a subject, comprising:administering to the subject a therapeutically effective dosage of apharmaceutical composition comprising an engineered cell, having anantibody covalently bound to a glycan moiety present on its surface. Inone embodiment, the cancer is breast cancer. In one embodiment, theantibody is Trastuzumab. In one embodiment, the cell is NK-92MI. In oneembodiment, more than one antibody is conjugated on the surface of thecell to target more than one cancer at the same time.

Embodiments of the instant disclosure further include a method of makingan engineered cell, comprising: making a fucose derivative or GDP-fucosederivative comprising a chemical or biological moiety covalently boundto a fucose or GDP-fucose; making the engineered cell by incubating acell with a composition comprising (a) the fucose derivative and/orGDP-Fucose derivative and (b) a fucosyltransferase enzyme. In anotherembodiment, disclosed herein is a method of making an engineered cell,comprising: generating CMP-sialic acid conjugated with an antibody(CS-IgG) by reacting an antibody bearing a TCO moiety with CS-Az-Tz orCS-Poc-Tz; and making the engineered cell by incubating a naturallyoccurring cell with a composition comprising (a) CS-IgG and (b) asialyltransferase. In one embodiment, the fucosyltransferase enzyme isα-1,3-fucosyltransferase. In one embodiment, theα-1,3-fucosyltransferase is H. pylori α-1,3-fucosyltransferase. In oneembodiment, the α-1,3-fucosyltransferase is recombinantly prepared. Inone embodiment, the sialyltransferase is selected from the groupconsisting of ST6Gal1, Pasteurella multocida α(2,3) sialyltransferaseM144D mutant (Pm2,3ST-M144D), and Photobacterium damsel α(2,6)sialyltransferase (Pd2,6ST). In one embodiment, the chemical orbiological moiety is selected from the group consisting of small drugmolecules, biomolecules, probe molecules, fluorophores, polynucleotides,polypeptides, and whole IgG, or combinations thereof. In one embodiment,the biomolecule is Trastuzumab. In one embodiment, the cell is an immunecell. In one embodiment, the cell is a primary human T cell or a naturalkiller (NK) cell. In one embodiment, the NK cell is NK-92MI cell. In oneembodiment, the T cell is a CD4+ cell, or a CD8+ T cells. In oneembodiment, the chemical or biological moiety is covalently attached viaa fucose derivative to LacNAc, a universal unit of N-glycans, on thesurface of the cell. In one embodiment, the fucose modified biomoleculeand/or GDP-fucose modified biomolecule comprises a fucose-alkyne and/orGDP-fucose azide. In one embodiment, the biomolecule-cell conjugate isfurther modified with a chemical or biological moiety using ligandaccelerated and biocompatible copper(I)-catalyzed alkyne-azidecycloaddition (CuAAC) reaction.

Various embodiments disclosed herein also include a one-pot method ofmaking an engineered cell adapted to attach a molecule on its surfacecomprising: preparing GDP-fucose analog by combining a fucose analogwith a mixture comprising ATP, GTP, L-fucokinase/GDP-fucosepyrophosphorylase (FKP), Mg²⁺ or Mn²⁺, and inorganic pyrophosphate(PPase); and making the engineered cell by adding a naturally occurringcell to a composition comprising the crude product from step (a) and H.pylori α-1,3-fucosyltransferase. In one embodiment, disclosed herein isa one-pot, one-step method of making an engineered cell adapted toattach a molecule on its surface comprising: incubating a cell with acomposition comprising a fucose analog, ATP, GTP,L-fucokinase/GDP-fucose pyrophosphorylase (FKP), Mg²⁺ or Mn²⁺, inorganicpyrophosphate (PPase) and H. pylori α-1,3-fucosyltransferase. In oneembodiment, the fucose analog comprises a fucose conjugated with achemical or biological moiety. In one embodiment, the fucose analogcomprises a fucose-alkyne. In one embodiment, the engineered cellcomprises a chemical or biological moiety attached to a glycan on thesurface of the cell. In one embodiment, the product from step (a) isfurther modified through a Copper-Catalyzed Azide-Alkyne Cycloaddition(CuACC) reaction to generate GDP-fucose analogs conjugated to a smallmolecule, polynucleotide, polypeptide, and/or antibody.

Further embodiments of the instant disclosure include a one pot in-situfucosylation strategy to convert cell surface LacNAc or SLacNAc into LeXor sLeX comprising: preparing GDP-fucose analogs by combining a fucoseanalog with a mixture comprising ATP, GTP, FKP, Mg²⁺ or Mn²⁺, andinorganic pyrophosphate (PPase); and converting cell surface LacNAc orSLacNAc into LeX or sLeX by adding a naturally occurring cell to acomposition comprising the crude product from step (a) and H. pyloriα-1,3-fucosyltransferase.

Various embodiments disclosed herein also include a kit comprising:GDP-fucose derivative and H. pylori α-1,3-fucosyltransferase.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousembodiments of the invention.

DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts, in accordance with embodiments herein, in situ one-potfucosylation reagents preparation and cell-surface reaction scheme.

FIG. 2 depicts, in accordance with embodiments herein, in situ one-potfucosylation of cultured Chinese Hamster Ovary (CHO) cells using crudeone-pot GDP-fucose.

FIG. 3 depicts, in accordance with embodiments herein, modification ofcultured Lec2 CHO cell surface using crude one-pot GDP-fucose-biotin.

FIG. 4 depicts, in accordance with embodiments herein, modification ofcultured Lec2 CHO cell surface using crude one-pot GDP-fucose-Cy3.

FIG. 5 depicts, in accordance with embodiments herein, modification ofcultured Lec2 CHO cell surface using crude one-pot GDP-fucose-tetrazine.

FIG. 6 depicts, in accordance with embodiments herein, modification ofcultured Lec2 CHO cell surface using crude one-pot GDP-fucose-BSA.

FIG. 7 depicts, in accordance with embodiments herein, modification ofcultured Lec2 CHO cell surface using crude one-pot GDP-fucose-antibody.

FIG. 8 depicts, in accordance with embodiments herein, modification ofcultured Lec2 CHO cell surface using crude one-pot GDP-fucose-Tz andsubsequent TCO-antibody.

FIG. 9 depicts, in accordance with embodiments herein, modification ofcultured wild type CHO cells surface using crude one-potGDP-fucose-biotin.

FIG. 10 depicts, in accordance with embodiments herein, modification ofactivated mouse CD8+ cells surface using crude one-potGDP-fucose-biotin.

FIG. 11 depicts, in accordance with embodiments herein, modification ofmouse dendritic cell (DC) surface using crude one-pot GDP-fucose-biotin.

FIG. 12 depicts, in accordance with embodiments herein, modification ofnaïve or activated human CD8+ T cell surface using crude one-potGDP-fucose-biotin.

FIG. 13 depicts, in accordance with embodiments herein, modification ofactivated mouse CD8+ T cell surface using crude one-pot GDP-fucose-Tzand then conjugated with TCO-mouse IgG.

FIG. 14 depicts, in accordance with embodiments herein, modification ofactivated mouse CD8+ T cell surface using crude one-pot GDP-fucose-Tzand then conjugated with TCO-mouse IgG and TCO-rat IgG together.

FIG. 15 depicts, in accordance with embodiments herein, installation ofanti-PD-L1 antibody on OT-1 T cell surface.

FIG. 16 depicts, in accordance with embodiments herein, installation ofanti-PD-L1 antibody on OT-1 T cell surface boost the adoptive celltherapy (ACT).

FIG. 17 depicts, in accordance with embodiments herein, in-situ one-potfucosylation of in vivo expanded mouse CD8+ T cells increase E-selectinbinding and suppress Gal-1 binding.

FIG. 18 depicts, in accordance with embodiments herein, in-situ one-potfucosylation of induced mouse regulatory T cells (iTreg) increaseE-selectin binding.

FIG. 19 depicts, in accordance with embodiments herein, in-situ one-potfucosylation of human T cells increases E-selectin binding.

FIG. 20 depicts, in accordance with embodiments herein, in-situ one-potfucosylation of human natural regulatory T (nTreg) cells increaseE-selectin binding and inhibit Gal-1 binding.

FIG. 21 depicts, in accordance with embodiments herein, in-situ one-potfucosylation of Her2 specific human CAR-T cells increases E-selectinbinding and inhibits Gal-1binding.

FIG. 22 depicts, in accordance with embodiments herein, in situ one-potfucosylation of in vitro differentiated mouse CD8 T cells significantlyenhanced E-selectin binding and decreased Gal-1 binding.

FIG. 23 depicts, in accordance with embodiments herein, in situ one-potfucosylation of in vitro differentiated mouse OT-1 CD8 T cells hadlittle impact on the expression of cell-surface markers.

FIG. 24 depicts, in accordance with embodiments herein, treatment ofB16-OVA-embeded mice with in vitro differentiated, glycan modified OT-1T cells significantly decreased tumor growth.

FIG. 25 depicts, in accordance with embodiments herein, treatment ofB16-OVA-embedded mice with in vivo differentiated, glycan modified OT-1T cells significantly decreased tumor growth and extended the life spanof the tumor embedded mice.

FIG. 26 depicts, in accordance with embodiments herein, in situ one-potfucosylation of in vitro differentiated mouse dendritic cells (DCs)significantly enhanced E-selectin binding and decreased Galectin-1binding.

FIG. 27 depicts, in accordance with embodiments herein, fucosylation ofantigen loaded DCs induces a significantly stronger anti-tumor immuneresponse.

FIG. 28 depicts, in accordance with embodiments herein, modification ofcultured Lec2 CHO cell surface using crude one-pot GDP-fucose-DNA-FAM.

FIG. 29 depicts, in accordance with embodiments herein, anti-humanE-selectin installed on cultured Lec2 CHO cell surface can bind to humanE-selectin.

FIG. 30 depicts, in accordance with embodiments herein, anti-mouse PD-L1installed on cultured Lec2 CHO cell surface can bind to mouse PD-L1

FIG. 31 depicts, in accordance with embodiments herein, anti-mouse CTLA4installed on cultured CHO cell surface can bind to mouse CTLA4 protein.

FIG. 32 depicts, in accordance with embodiments herein, anti-mouse CTLA4installed on mouse dendritic cell surface can bind to mouse CTLA4protein.

FIG. 33 depicts, in accordance with embodiments herein, mouse IgGinstalled on OT-1 CD8 T cell surface can stay on surface for more than24 hours.

FIG. 34 depicts, in accordance with embodiments herein, conjugate OT-1CD8 T cell with mouse IgG antibodies does not affect the proliferationrate of the modified cells.

FIG. 35 depicts, in accordance with embodiments herein, anti-mouse PD-L1installed on OT-1 CD8 T cell surface can stay on cell surface for morethan 24 hours.

FIG. 36 depicts, in accordance with embodiments herein, conjugate OT-1CD8 T cell with anti-PD-L1 antibodies doesn’t affect the proliferationrate of modified cells.

FIG. 37 depicts, in accordance with embodiments herein, anti-mouse PD-L1installed on OT-1 CD8 T cell surface can bind to mouse PD-L1.

FIG. 38 depicts, in accordance with embodiments herein, anti-mouse PD-L1installed on OT-1 CD8 T cell surface can improve T cells’ killingfunction on cancer cells.

FIG. 39 depicts, in accordance with embodiments herein, a one-stepenzymatic fucosylation based strategy for cell-surface engineering. (A)Two conventionally used cell-surface engineering approaches. Metabolicengineering is used to install a reaction handle (X) onto the cellsurface, which can react with a complementary handle (Y) on a moleculeof interest. Genetic engineering is powerful and robust, which couldinstall functional molecules and reaction handles on cell surface. Thesetwo approaches usually have multistep and last several days. (B) Anenzymatic glycoengineering approach capable of transferring a variety offunctional molecules to the cell surface in one-step. The reactionbetween cell surface LacNAc/sialylLacNAc and GDP-Fuc derivatives isenabled by a α1,3FucT from H. pylori, which could tolerate modificationsas large as whole IgG at the C6 position of the fucose. (C) One-potsynthesis protocol for GF-A1 and GF-Az derivatives is described. Shownfunctionalities (Z) include bioorthogonal handles (tetrazine, Tz),biophysical probes (biotin, Cy3), and biomaterials (ssDNA).

FIG. 40 depicts, in accordance with embodiments herein, enzymatictransfer of IgG to the cell surface of Lec2CHO cells. (A) Schematicrepresentation of GDP-Fuc modified IgG (GF-IgG) synthesis. Chemicalstructures of bioorthogonal handles and linkers are shown. (B) Scheme ofusing FucT to transfer GF-IgG to CHO Lec2 cell surface. (C) Flowcytometry analysis of Lec2 cells treated with enzyme FT, substratesGF-rIgG or both. (D) Titration of GF-rIgG concentrations (from 0.005mg/ml to 0.2 mg/ml) in enzymatic transfer. Each reaction used sameamount of FT and was left in room temperature for 30 min. Mean ± SD(error bars), representative graph from three independent experiments.(E) Time course of enzymatic transfer of GF-rIgG to Lec2 cells on ice.Reaction at 37° C. was a maximum labeling control. (F) Flow cytometryanalysis of Lec2 cells viability before and after IgG labeling. (G) CHOLec8 cells without LacNAc expression was compared with Lec2 cells inenzymatic IgG transfer as a negative control. Mean ± SD (error bars),representative graph from three independent experiments. (H) Confocalmicroscopy images of Lec2 cells treated with or without FT whenincubated with Alexa Fluor 647 labeled GF-rIgG. Nuclei were stained withHoechst 33342. Scale bar: 2 µm. (I) Flow cytometry analysis of Lec2cells labeled with rIgG and mIgG simultaneously.

FIG. 41 depicts, in accordance with embodiments herein, enzymatictransfer of α-E-selectin to primary human T cells. (A) Schematicillustration of modified (1) and natural (2) process of human T cellbinding and transmigration on HUVEC cells. (B) FT mediated GF-α-hE-Sellabeling on primary human T cells from three different donors. Mean ± SD(error bars), representative graph from three independent experiments.(C) Binding of human E-selectin-Fc chimera on unlabeled T cells and Tcells labeled with mIgG or α-hE-Sel. Mean ± SD (error bars). (D)Analysis of flow chamber assay on human E-selectin-Fc coated slidesunder shear stress conditions. T cells labeled with mIgG or α-hE-Selwere compared with unlabeled T cells. Cell numbers (cells/mm²) werequantified in ImageJ. Mean ± SD (error bars). (E,F) Quantitativeanalysis and fluorescent microscopy images of human T cells binding onHUVEC. Human T cells were stained with CFSE (green) and HUVEC cells werestained with Hoechst 33342 (blue) and DiD (red). HUVEC were pretreatedwith TNF-α if indicated. Mean ± SD (error bars), representative graphfrom three independent experiments. Scale bar: 50 µm. (G) Analysis oftransmigration assay on HUVEC. Migrated T cells (LeukoTracker, Green) inthe bottom layer were quantified by fluorescence signal. In all figures:ns, P >0.05; *P < 0.05; **P < 0.01; ****P < 0.0001.

FIG. 42 depicts, in accordance with embodiments herein, construction ofHerceptin and NK-92MI cells conjugates for HER2+ cancer therapy. (A)NK-92MI-Hercetin conjugates could specifically bind to HER2+ cancercells and hence have enhanced killing effect due to proximity. (B)Analysis of specific HER2 antigen binding on NK-92MI-Hereceptinconjugates. Mean ± SD (error bars). (C,D) Flow cytometry analysis andfluorescent microscopy images of specific binding betweenNK-92MI-Herceptin and BT474 (HER2+). NK-92MI cells are stained withCellTracker Orange (red) and BT474 cells are stained with CellTrackerGreen (green). Shown are merged channels of fluorescence and phasecontrast. Green fields are clusters of BT474 cells. Scale bar: 50 µm.(E) LDH release assay of quantifying cell-mediated cytotoxicity ofNK-92MI cells against BT474 cells. NK-92MI-Herceptin was compared withparental NK-92MI with or without additional added free Herceptin (5µg/ml). NK-92MI-hIgG was used as a negative control. Mean ± SD (errorbars), representative graph from three independent experiments. (F)Comparison of NK-92MI and NK-92MI-Herceptin in killing different cancercell lines with or without HER2 expression. Mean ± SD (error bars),representative graph from three independent experiments. (G) Killingactivity of NK-92MI-Herceptin in different GF-Herceptin concentrationsfor enzymatic transfer. Mean ± SD (error bars). (H) Comparison ofNK-92MI and NK-92MI-Herceptin in killing BT474 under different effectorto target cell ratios. Mean ± SD (error bars). (I) Herceptin and α-EGFRdual labeled NK-92MI cells were compared with NK-92MI-Herceptin andNK-92MI-α-EGFR in killing HER2+/EGFR+ SKOV3 cancer cells. Mean ± SD(error bars). (J) Comparison of non-irradiated and irradiated (6 Gy)NK-92MI cells in killing BT474 cells. (K) In vivo antitumor activity ofNK-92MI-Herceptin. NSG mice were intravenously injected with 0.5 millionMDA-MB-435/HER2+/F-luc cells. Then, animals were treated once by i.v.injection of 5 million NK-92MI or NK-92MI-Herceptin cells at day 1 aftertumor cell injection. Control mice received HBSS. Six days after tumorchallenge, mice were injected with i.p. with D-luciferin and imaged byIVIS system. Tumor size of individual mice and mean values ± SD areshown; n = 10. Representative images are also shown. In all figures: ns,P >0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

FIG. 43 depicts, in accordance with embodiments herein, enzymatictransfer of α-PD-L1 to OT-1 T cells for enhanced T cell activation inspecific killing. (A) Scheme of the interaction between OT-1 T cells(TCR-T) and B16-OVA melanoma cells. MHC complex on B16-OVA present OVAantigen to specific TCR on OT-1 T cells to induce activation, whilePD-L1 on B16-OVA interact with PD-1 on OT-1 T cells inhibit theactivation signal. (B) Scheme of the blockade of PD-1/PD-L1 pathway viaα-PD-L1 conjugated on OT-1 T cell surfaces. The in situ blockade couldenhance T cell activation and killing on cancer cells. (C) Analysis ofPD-L1 antigen binding on OT-1 T cells under different treatment. Mean ±SD (error bars). (D) Analysis of PD-L1 antigen binding on differentGF-α-PD-L1 concentrations in enzymatic transfer. Mean ± SD (error bars).(E) Assay of quantifying cell-mediated cytotoxicity of OT-1 Tcell-α-PD-L1 conjugates on B16-OVA cells. Mean ± SD (error bars),representative graph from three independent experiments. OT-1 T cellsconjugated with rIgG and P14 T cells conjugated with α-PD-L1 were shownas negative control. (F) Comparison of OT-1 T cells and OT-1 Tcell-α-PD-L1 in killing B16-OVA under different effector to target cellratios. Mean ± SD (error bars). (G) Killing activity of OT-1 T cells andOT-1 T cell-α-PD-L1 in different GF-α-PD-L1 concentrations for enzymatictransfer. Mean ± SD (error bars). (H) IFN-γ ELISA of OT-1 T cells mixedwith B16-OVA under different treatment. P14 T cell conjugated withα-PD-L1 was shown as negative control. Only B16-OVA was background. Mean± SD (error bars), representative graph from three independentexperiments. (I) Microscopy images of OT-1 T cells killing B16-OVA withor without α-PD-L1 labeling. Blue arrow indicates less cancer cells andpurple arrow indicates bigger cluster of T cells. Scale bar: 50 µm. (J)Analysis of OT-1 T cell proliferation after the activation mediated byB16-OVA through CFSE dilution. Mean ± SD (error bars). (K) Competitionassay of comparing T cells frequency in blood, draining lymph nodes, andtumor with or without α-PD-L1 labeling. OT-1 T cells with CD45.1 orThy1.1 congenic marker were labeled with α-PD-L1, which were later mixedwith unlabeled OT-1 T cells with the other congenic marker. The OT-1 Tcells mixture was i.v. injected into B16-OVA challenged mice andanalyzed after 48 hours expansion. Relative ratio of individual mice andmean values ± SD are shown; n = 3. In all figures: ns, P >0.05; **P <0.01; ***P < 0.001; ****P < 0.0001.

FIG. 44 depicts, in accordance with embodiments herein, one-stepenzymatic labeling is more efficient and biocompatible than two-step.(A) Scheme of one-step and two-step labeling system. In one steplabeling system, one-pot product of GF-Al-biotin was directlytransferred to LacNAc epitope on Lec2 cell surface using α1,3-FucT. Incontrast, the two-step labeling involved the first step of enzymatictransfer of GF-A1 and a followed step of CuAAC reaction between surfacealkyne and azide-biotin probe. (B) Flow cytometry analysis ofbiocompatibility in one-step or two-step labeling process. Lec2 cellsafter reaction were stained with DAPI. (C) Comparison of efficiency inone-step and two-step labeling process described in (A). Cells werestained by streptavidin-APC.

FIG. 45 depicts, in accordance with embodiments herein, chemicalstructures of GDP-Fuc derivatives including GF-Al-Biotin, GF-Al-Cy3,GF-Al-Tz and GF-Az-Tz.

FIG. 46 depicts, in accordance with embodiments herein, one-stepenzymatic transfer of one-pot GDP-Fuc derivatives to Lec2 cells. (A)Scheme of transferring one-pot GDP-Fuc derivatives using α1,3-FucT toLacNAc epitope on Lec2 CHO cell surface. (B) Lec2 cells were treatedwith FT and GF-Al-Cy3, FT alone, or GF-Al-Cy3 alone, or untreated. Thencells were analyzed by flow cytometry, which only showed successfullabeling when treated with both FT and GF-Al-Cy3. (C) Lec2 cells weretreated with FT and GF-Az-Tz, FT alone, or GF-Az-Tz alone, or untreated.Then the cells were reacted with TCO-Cy5 and analyzed by flow cytometry.Clear labeling is only shown in the group treated with FT and GF-Az-Tz.(D) GF-Al-Tz and GF-Az-Tz were compared in the FT mediated transferreaction on Lec2 cells. Cells were reacted with TCO-Cy5 after enzymaticreaction and analyzed by flow cytometry. GF-Az-Tz group showed strongersignal than GF-Al-Tz. (E) Lec2 cells were treated with FT andGF-Az-ssDNA-FAM, or GF-Az-ssDNA-FAM alone, or untreated. Then the cellswere analyzed by flow cytometry. The group treated with FT andGF-Az-ssDNA-FAM has the strongest FAM signal.

FIG. 47 depicts, in accordance with embodiments herein, molecular modelsof GDP-fucose (A) and GF-Az-Tz (B) in the active site of α1,3-FucT.Images are created from the previous reported crystal structure (PDB:2NZY) of HP-FucT and GDP-fucose complex in PyMOL.

FIG. 48 depicts, in accordance with embodiments herein, the blockade ofLacNAc epitope inhibits FT mediated GF-rIgG transfer to Lec2 cellsurface. Lec2 cells were treated with FT and GDP-Fuc first, which couldoccupy almost all of the LacNAc epitope. After that, blocked cells weretreated with FT and GF-rIgG. rIgG labeling were analyzed by flowcytometry. Compared to direct GF-rIgG transfer, the blocked Lec2 have asignificantly reduced rIgG signal. Error bars, mean values ± SD. ****P <0.0001.

FIG. 49 depicts, in accordance with embodiments herein, dual labeling oftwo different IgG molecules on Lec2 cells. Lec2 cells were treated withGF-mIgG, GF-rIgG and FT. GF-mIgG and GF-rIgG treated was shown as anegative control. The labeled cells were stained with anti-mIgG andanti-rIgG fluorescent antibodies and analyzed by flow cytometry.

FIG. 50 depicts, in accordance with embodiments herein, phenotype ofhuman T cells before and after mIgG labeling. (A) Human PBMC wereactivated by anti-CD3/CD28 antibody and expanded in vitro for about twoweeks. The cells were stained with anti-CD3, anti-CD45, anti-CD4 andanti-CD8 fluorenscent antibodies, and analyzed by flow cytometry. (B)Activated human T cells were treated with GF-IgG and FT. Labeled orunlabeled cells were stained with anti-CD4, anti-CD8, anti-CD25,anti-CD45RO, anti-CD44 and anti-CD62L fluorescent antibodies andanalyzed by flow cytometry. Naive T cells were used as control.

FIG. 51 depicts, in accordance with embodiments herein, transferring IgGmolecules onto human T cells. (A) LacNAc on Human PBMC was labeled byGF-Biotin on different days after activation. Labeled cells were stainedwith streptavidin-APC, anti-CD4 and anti-CD8 fluorescent antibodies, andanalyzed by flow cytometry. (B) Human T cells were treated with FT andGF-α-hE-Sel, or GF-α-hE-Sel alone, or untreated. The cells were stainedwith anti-mIgG fluorescent antibody and analyzed by flow cytometry. (C)Human T cells were treated with FT and GF-mIgG, or GF-mIgG alone, oruntreated. The cells were stained with anti-mIgG fluorescent antibodyand analyzed by flow cytometry. (D) Human T cells labeled with α-hE-Selwere stained with anti-mIgG fluorescent antibody and analyzed by flowcytometry at different time points after labeling. (E) Human T cellswith or without mIgG labeling were cultured in T cell media and livecells in each group were counted on different days after labeling. (F)Human T cells with different modifications were incubated withE-selectin-Fc protein and then stained with anti-hFc fluorescentantibody for FACS analysis. Error bars, mean values ± SD. In allfigures: ****P < 0.0001.

FIG. 52 depicts, in accordance with embodiments herein, transferring IgGmolecules onto NK-92MI cells. (A) NK-92MI cells were treated withGF-hIgG and FT, or GF-hIgG alone, or untreated. The cells were stainedwith anti-hFc fluorescent antibody and analyzed by flow cytometry. (B)NK-92MI cells were treated with GF-Herceptin and FT, or GF-Herceptinalone, or untreated. The cells were stained with anti-hFc fluorescentantibody and analyzed by flow cytometry. (C) NK-92MI cells conjugatedwith Herceptin were stained with anti-hFc fluorescent antibody andanalyzed by flow cytometry at different time points post labeling. (D)NK-92MI cells with different modifications were then incubated withHER2-His6 protein and then stained with anti-His6 fluorescent antibodyfor FACS analysis. (E) NK-92MI cells conjugated with or withoutHerceptin were co-cultured with BT474 at a ratio of 5:1 (Effector :Target) for 4 h. Granzyme B secretion in culture supernatant wasanalyzed by ELISA. Error bars, mean values ± SD. In all figures: ns,P >0.05; ****P < 0.0001.

FIG. 53 depicts, in accordance with embodiments herein, titration ofGF-Herceptin concentrations in enzymatic transfer. NK-92MI cells weretreated with different concentrations of GF-Herceptin in a standardlabeling condition. Labeled cells were directly stained with anti-hFcfluorescent antibody (A), or incubated with HER2-His6 and then stainedwith anti-His6 fluorescent antibody (B) before flow cytometry analysis.

FIG. 54 depicts, in accordance with embodiments herein, dual antibodylabeling on NK-92MI cells. NK-92MI cells were treated with FT andGF-Herceptin, and then treated with FT and GF-α-EGFR. Labeled cells werestained with anti-hFc fluorescent antibody (A), or stained withanti-mIgG fluorescent antibody (B) before flow cytometry analysis.

FIG. 55 depicts, in accordance with embodiments herein, growth andcytotoxic activity of NK-92MI cells upon γ-irradiation. NK-92MI cellswere received with or without 6 Gy gamma irradiations before enzymaticreaction. Labeled and unlabeled NK-92MI cells were cultured for anotherthree days. (A) Viable cells were counted by flow cytometry at differenttime points after irradiation and labeling. (B) Herceptin labeled cellswere also stained with anti-hFc fluorescent antibody and analyzed byflow cytometry at different time points post labeling. Irradiated cellshas a slower decay of Herceptin labeling. Error bars, mean values ± SD.In all figures: ns, P >0.05; ***P < 0.001; ****P < 0.0001.

FIG. 56 depicts, in accordance with embodiments herein, images ofNK-92MI-Herceptin showing enhanced antitumor activity in mice. Shown areimages of 30 mice in three groups. The data is summarized and analyzedin FIG. 4K.

FIG. 57 depicts, in accordance with embodiments herein, enzymatictransfer of rIgG to OT-1 CD8+ T cells. (A-B) Spleenocytes from OT-1 micewere activated by OVA peptides and in vitro expanded by adding IL2 orIL7/IL15. After four days expansion, the cells were stained withanti-CD3 and anti-CD8 fluorescent antibodies and analyzed by flowcytometry (A). The cells were also treated with GF-Al-Biotin and FT, andthen stained with streptavidin-APC and analyzed by flow cytometry totrack LacNAc level on different days after T cell activation (B). (C)OT-1 T cells were treated with GF-rIgG and FT, or GF-rIgG alone, oruntreated. Then the cells were stained with anti-rIgG fluorescentantibody and analyzed by flow cytometry. (D) OT-1+/-CD45.1+/- T cellswere first labeled with CFSE and then treated with GF-rIgG and FucT, oruntreated. After fucosylation, the cells were co-cultured with OVApeptide pulsed WT B6 spleenocytes for 48 h and T cell proliferation wasanalyzed by flow cytometry. Error bars, mean values ± SD. In allfigures: ns, P >0.05; ****P < 0.0001.

FIG. 58 depicts, in accordance with embodiments herein, transferringα-PD-L1 onto OT-1 CD8+ T cells. (A) OT-1 CD8+ T cells were treated withGF-α-PD-L1 and FT, or GF-α-PD-L1 alone, or untreated. The cells werethen stained with anti-rIgG fluorescent antibody and analyzed by flowcytometry. (B-C) CD8+ T cells from P14 mice were treated with GF-α-PD-L1and FT, or GF-α-PD-L1 alone, or untreated. The cells were stained withanti-rIgG fluorescent antibody (B), or incubated with PD-L1-hFc firstand then stained with anti-hFc fluorescent antibody (C). After staining,these cells were analyzed by flow cytometry. (D) OT-1 T cells werelabeled under different concentrations of GF-α-PD-L1 with or without FT.After labeling, cells were stained with anti-rIgG fluorescent antibodyand analyzed by flow cytometry. (E) OT-1 T cells were treated withGF-α-PD-L1 and FT, or GF-α-PD-L1 alone, or untreated. The cells werethen stained with anti-rIgG fluorescent antibody and analyzed by flowcytometry at different time points after reaction. Error bars, meanvalues ± SD. In all figures: ns, P >0.05; ****P < 0.0001.

FIG. 59 depicts, in accordance with embodiments herein, transferringα-PD-L1 onto OT-1 T cell surface enhance the cytokine secretion duringkilling cancer cells. (A) OT-1 T cells and P14 T cells were treated withGF-α-PD-L1 and FT, or GF-rIgG and FT, or untreated. T cells were thenco-cultured with B16-OVA cells for 9 hours. TNF-α concentrations inculture supernatant were quantified via ELISA kit. (B-C) OT-1 T cellswere treated under different concentrations of GF-α-PD-L1 with orwithout FT, and then co-cultured with B16-OVA cells for 9 hours. TNF-αand IFN-γ in culture supernatant were analyzed by ELISA. Error bars,mean values ± SD. In all figures: ns, P >0.05; ***P < 0.001; ****P <0.0001.

FIG. 60 depicts, in accordance with embodiments herein, transferringα-PD-L1 onto T cell surface increase the size of T cell cluster and Tcell proliferation during killing. (A) OT-1 T cells and P14 T cells weretreated with GF-α-PD-L1 and FT, or GF-rIgG and FT, or untreated. Thecells were then co-cultured with B16-OVA cells for 20 hours. T cellcluster during killing were imaged using microscope. Scale bar: 50 µm.(B) OT-1 T cells were stained with CFSE and treated with GF-α-PD-L1 andFT, or GF-rIgG and FT, or untreated. Then the cells were cultured withor without B16-OVA cells for 72 hours. Proliferations of OT-1 T cellswere analyzed through CFSE signal dilution.

FIG. 61 depicts, in accordance with embodiments herein, in vivoantitumor activity of NK-92MI-Herceptin in intraperitoneal cancer model.NSG mice were intraperitoneally injected with 1 millionMDA-MB-435/HER2+/F-luc cells. Then, animals were treated once by i.v.injection of HBSS, 5 million NK-92MI or NK-92MI-Herceptin cells at day 5and day 10 after tumor cell injection. On day 4, day 12, day 17 aftertumor challenge, mice were injected with i.p. with D-luciferin andimaged by IVIS system. (A) Representative images on day 12 after tumorinoculation showing enhanced antitumor activity in mice in Herceptinlabeled group. (B) Luciferin signal on day 4, day 12 and day17 wereanalyzed and calculated. (C) Luciferin signal on day 12 were analyzedand compared. (D) Luciferin signal on day 17 were analyzed and compared.Tumor size of individual mice and mean values ± SD are shown; n = 8.Representative images are also shown. In all figures: ns, P >0.05; *P <0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

FIG. 62 depicts, in accordance with embodiments herein, another AntibodyCell Conjugate (ACC) strategy. (A), Flow cytometry analysis of NK-92cells treated with GF-16A (anti-MUC1), or GF-16A and FT. (B), Titrationof GF-16A concentrations ranging from 0.05 mg/ml to 0.2 mg/ml in thereaction buffer; each reaction used the same amount of FT and proceededat room temperature for 30 min. (C) and (D), Flow cytometry analysis ofMUC1 expression on MDA-MB-435 and SKBR3 cancer cells. (E) and (F),Luciferase assay of quantifying cell-mediated cytotoxicity of NK-92MIcells against SKBR3-luc and MDA-MB-435-luc cells; 16A-NK-92MI conjugateswere compared with parental NK-92MI with or without additional addedfree 16 A (5 µg/ml). 16 A and NK-92MI cells only treated with GF-16A inlabeling were used as negative controls. Mean ± SD (error bars),representative graph from three independent experiments. ns, P > 0.05;**P <0.01.

FIG. 63 depicts, in accordance with embodiments herein, enzymaticbio-macromolecule transfer using a sialylation strategy. (A),Sialyltransferase enables enzymatic transferring a variety of functionalmolecules to the surfaces of cells in one step. (B), Chemical structuresof CMP-sialic acid-azide-tetrazine (CS-Az-Tz) and CMP-sialicacid-propargyl carbamate-tetrazine (CS-Poc-Tz). (C), Schematicrepresentation of the synthesis of a CMP-Sialic acid conjugated IgG(CS-IgG). (D), Workflow of the ST-catalyzed transfer of CS-IgG to thesurface of NK-92MI cells. (Three different sialyltransferases weretested here). (E), Flow cytometry analysis of NK-92MI cells treated withenzyme STs, substrates CS-Az-mIgG, or both. (F), Flow cytometry analysisof NK-92MI cells treated with enzyme STs, substrates CS-Poc-mIgG, orboth.

DETAILED DESCRIPTION

All references, publications, and patents cited herein are incorporatedby reference in their entirety as though they are fully set forth.Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Hornyak, et al., Introductionto Nanoscience and Nanotechnology, CRC Press (2008); Singleton et al.,Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley &Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions,Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013);and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th ed.,Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012),provide one skilled in the art with a general guide to many of the termsused in the present application. One skilled in the art will recognizemany methods and materials similar or equivalent to those describedherein, which could be used in the practice of the present invention.Indeed, the present invention is in no way limited to the methods andmaterials described.

The term “fucosyltransferase,” as used herein, refers to an enzyme thattransfers a fucose sugar from a GDP-fucose (guanosinediphosphate-fucose) donor substrate to an acceptor substrate. Theacceptor substrate can be another sugar such as the transfer of a fucoseto a core GlcNAc (N-acetylglucosamine) sugar as in the case of N-linkedglycosylation, or to a protein, as in the case of O-linked glycosylationproduced by O-fucosyltransferase.

The terms “peptide,” “polypeptide” and “protein” are usedinterchangeably to refer to an isolated polymer of amino acid residues,and are not limited to a minimum length unless otherwise defined.Peptides, oligopeptides, dimers, multimers, and the like, are alsocomposed of linearly arranged amino acids linked by peptide bonds, andwhether produced biologically and isolated from the natural environment,produced using recombinant technology, or produced syntheticallytypically using naturally occurring amino acids. In some aspects, thepolypeptide or protein is a “modified polypeptide” comprisingnon-naturally occurring amino acids. In some aspects, the polypeptidescomprise a combination of naturally occurring and non-naturallyoccurring amino acids, and in some embodiments, the peptides compriseonly non-naturally occurring amino acids. The term “peptide” as usedherein encompasses native peptides (either degradation products,synthetically synthesized peptides or recombinant peptides) andpeptidomimetics (typically, synthetically synthesized peptides), as wellas peptoids and semipeptoids which are peptide analogs, which may have,for example, modifications rendering the peptides more stable while in abody or more capable of penetrating into cells. Such modificationsinclude, but are not limited to, N-terminus modification, C-terminusmodification, peptide bond modification, backbone modification, and/orside chain modification.

The term “antibody” as used herein contemplates a polypeptide or aprotein complex that specifically binds an epitope of an antigen ormimetope thereof. An antibody includes an intact antibody, or a bindingfragment thereof that competes with the intact antibody for specificbinding and includes chimeric, humanized, fully human, and bispecificantibodies. Binding fragments include, but are not limited to, Fab,Fab′, F(ab′)2, Fv, and single-chain antibodies. In some embodiments, anantibody is referred to as an immunoglobulin and include the variousclasses and isotypes, such as IgA (IgA1 and IgA2), IgD, IgE, IgM, andIgG (IgG1, IgG3 and IgG4) etc. in some embodiments the term “antibody”as used herein refers to polyclonal and monoclonal antibodies andfunctional fragments thereof. An antibody includes modified orderivatised antibody variants that retain the ability to specificallybind an epitope. Antibodies are capable of selectively binding to atarget antigen or epitope. Antibodies may include, but are not limitedto polyclonal antibodies, monoclonal antibodies (mAbs), humanized andother chimeric antibodies, single chain antibodies (scFvs), Fabfragments, F(ab′)2 fragments and disulfide-linked Fvs (sdFv) fragments.In some embodiments, the antibody is from any origin, such as mouse orhuman, including a chimeric antibody thereof. In some embodiments, theantibody is humanized.

Examples of antibodies include, but are not limited to, 3F8, 8H9,Abagovomab, Abciximab (ReoPro), Abituzumab, Abrilumab, Actoxumab,Adalimumab (Humira), Adecatumumab, Aducanumab, Afasevikumab, Afelimomab,Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab (Lemtrada, Campath),Alirocumab (Praluent), Altumomab pentetate (Hybri-ceaker), Amatuximab,Anatumomab mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab,Apolizumab, Arcitumomab (CEA-Scan), Ascrinvacumab, Aselizumab,Atezolizumab (Tecentriq), Atinumab, Atorolimumab, Avelumab (Bavencio),Bapineuzumab, Basiliximab (Simulect), Bavituximab, Bectumomab(LymphoScan), Begelomab, Belimumab (Benlysta), Benralizumab,Bertilimumab, Besilesomab (Scintimun), Bevacizumab (Avastin),Bezlotoxumab (Zinplava), Biciromab (FibriScint), Bimagrumab,Bimekizumab, Bivatuzumab mertansine, Bleselumab, Blinatumomab(Blincyto), Blontuvetmab (Blontress), Blosozumab, Bococizumab,Brazikumab, Brentuximab vedotin (Adcentris), Briakinumab, Brodalumab(Siliz), Brolucizumab, Brontictuzumab, Burosumab, Cabiralizumab,Canakinumab (Ilaris), Cantuzumab mertansine, Cantuzumab ravtansine,Caplacizumab, Capromab pendetide (Prostascint), Carlumab, Carotuximab,Catumaxomab (Removab), cBR96-doxorubicin immunoconjugate, Cedelizumab,Cergutuzumab amunaleukin, Certolizumab pegol (Cimzia), Cetuximab(Erbitux), Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab,Clivatuzumab tetraxetan (hPAM4-Cide), Codrituzumab, Coltuximabravtansine, Conatumumab, Concizumab, Crenezumab, Crotedumab, CR6261,Dacetuzumab, Daclizumab (Zenapax), Dalotuzumab, Dapirolizumab pegol,Daratumumab (Darzalex), Dectrekumab, Demcizumab, Denintuzumab mafodotin,Denosumab (Prolia), Depatuxizumab mafodotin, Derlotuximab biotin,Detumomab, Dinutuximab (Unituxin), Diridavumab, Domagrozumab, Dorlimomabaritox, Drozitumab, Duligotumab, Dupilumab (Dupixent), Durvalumab(Imfinzi), Dusigitumab, Ecromeximab, Eculizumab (Soliris), Edobacomab,Edrecolomab (Panorex), Efalizumab (Raptiva), Efungumab (Mycograb),ldelumab, Elgemtumab, Elotuzumab (Repatha), Elsilimomab, Emactuzumab,Emibetuzumab, Emicizumab, Enavatuzumab, Enforfumab vedotin, Enlimomabpegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan, Epratuzumab, Erenumab, Erlizumab, Ertumaxomab (Rexomun),Etaracizumab (Abegrin), Etrolizumab, Evinacumab, Evolocumab (Repatha),Exbivirumab, Fanolesomab (NeutroSpec), Faralimomab, Farletuzumab,Fasinumab, FBTA05 (Lymphomun), Felvizumab, Fezakinumab, Fibatuzumab,Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab,Fontolizumab (HuZAF), Foralumab, Foravirumab, Fresolimumab, Fulranumab,Futuximab, Galcanezumab, Galiximab, Ganitumab, Gantenerumab,Gavilimomab, Gemtuzumab ozogamicin (Mylotarg), Gevokizumab, Girentuximab(Rencarex), Glembatumumab vedotin, Golimumab (Simponi), Gomiliximab,Guselkumab, Ibalizumab, Ibritumomab tiuxetan (Zevalin), Icrucumab,Idarucizumab (Praxbind), Igovomab (Indimacis-125), IMAB362, Imalumab,Imciromab (Myoscint), Imgatuzumab, Inclacumab, Indatuximab ravtansine,Indusatumab vedotin, Inebilizumab, Infliximab (Remicade), Intetumumab,Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Isatuximab,Itolizumab, Ixekizumab (Taltz), Keliximab, Labetuzumab (CEA-Cide),Lampalizumab, Lanadelumab, Landogrozumab, Laprituximab emtansine,Lebrikizumab, Lemalesomab, Lendalizumab, Lenzilumab, Lerdelimumab,Lexatumumab, Libivirumab, Lifastuzumab vedotin, Ligelizumab, Lilotomabsatetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab,Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Lumiliximab,Lumretuzumab, MABp1 (Xilonix), Mapatumumab, Margetuximab, Maslimomab,Mavrilimumab, Matuzumab, Mepolizumab (Bosatria), Metelimumab,Milatuzumab, Minretumomab, Mirvetuximab soravtansine, Mitumomab,Mogamulizumab, Monalizumab, Morolimumab, Motavizumab (Numax),Moxetumomab pasudotox, Muromonab-CD3 (Orthoclone OKT3), Nacolomabtafenatox, Namilumab, Naptumomab estafenatox, Naratuximab emtansine,Narnatumab, Natalizumab (Tysabri), Navicixizumab, Navivumab, Nebacumab,Necitumumab (Portrazza), Nemolizumab, Nerelimomab, Nesvacumab,Nimotuzumab (Theracim, Theraloc), Nivolumab (Opdivo), Nofetumomabmerpentan (Verluma), Obiltoxaximab, Obinutuzumab (Gazyva), Ocaratuzumab,Ocrelizumab (Ocrevus), Odulimomab, Ofatumumab (Arzerra), Olaratumab(Lartruvo), Olokizumab, Omalizumab (Xolair), Onartuzumab, Ontuxizumab,Opicinumab, Oportuzumab monatox, Oregovomab (OvaRex), Orticumab,Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab,Pagibaximab, Palivizumab (Synagis, Abbosynagis), Pamrevlumab,Panitumumab (Vectibix), Pankomab, Panobacumab, Parsatuzumab,Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab(Keytruda), Pemtumomab (Theragyn), Perakizumab, Pertuzumab (Omnitarg),Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab,Plozalizumab, Pogalizumab, Polatuzumab vedotin, Ponezumab, Prezalizumab,Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab,Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab (Cyramza),Ranibizumab (Lucentis), Raxibacumab, Refanezumab, Regavirumab,Reslizumab, Rilotumumab, Rinucumab, Risankizumab, Rituximab (MabThera,Rituxan), Rivabazumab pegol, Robatumumab, Roledumab, Romosozumab,Rontalizumab, Rovalpituzumab tesirine, Rovelizumab (LeukArrest),Ruplizumab (Antova), Sacituzumab govitecan, Samalizumab, Sapelizumab,Sarilumab, Satumomab pendetide, Secukinumab (Cosentyx), Seribantumab,Setoxaximab, Sevirumab, Sibrotuzumab, SGN-CD19A, SGN-CD33A, Sifalimumab,Siltuximab (Sylvant), Simtuzumab, Siplizumab, Sirukumab, Sofituzumabvedotin, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab,Sulesomab (LeukoScan), Suvizumab, Tabalumab, Tacatuzumab tetraxetan(AFP-Cide), Tadocizumab, Talizumab, Tamtuvetmab (Tactress), Tanezumab,Taplitumomab paptox, Tarextumab, Tefibazumab (Aurexis), Telimomabaritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab,Tetulomab, Tezepelumab, TGN1412, Ticilimumab (tremelimumab),Tildrakizumab, Tigatuzumab, Timolumab, Tisotumab vedotin, TNX-650,Tocilizumab (Actemra, RoActemra), Toralizumab, Tosatoxumab, Tositumomab(Bexxar), Tovetumab, Tralokinumab, Trastuzumab (Herceptin), Trastuzumabemtansine (Kadcyla), TRBS07 (Ektomab), Tregalizumab, Tremelimumab,Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab,Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab (Stelara), Utomilumab,Vadastuximab talirine, Vandortuzumab vedotin, Vantictumab, Vanucizumab,Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab (Entyvio), Veltuzumab,Vepalimomab, Vesencumab, Visilizumab (Nuvion), Vobarilizumab,Volociximab, Vorsetuzumab mafodotin, Votumumab (HumaSPECT), Xentuzumab,Zalutumumab (HuMax-EGFr), Zanolimumab (HuMax-CD4), Zatuximab,Ziralimumab, and Zolimomab. In one preferred embodiment, the antibody isTrastuzumab (Herceptin).

The term “antibody” as used herein also contemplates the biosimilar orsecond generation version of the monoclonal antibodies described herein.

The term “polynucleotide,” or “nucleotide” as used herein, refergenerally to linear polymers of natural or modified nucleosides,including deoxyribonucleosides, ribonucleosides, alpha-anomeric formsthereof, and the like, usually linked by phosphodiester bonds or analogsthereof ranging in size from a few monomeric units, e.g. 2-4, to severalhundreds of monomeric units. When a polynucleotide is represented by asequence of letters, such as “ATGCCTG,” it will be understood that thenucleotides are in 5′->3′ order from left to right. Polynucleotide asused herein also includes abasic, sugar-phosphate or sugar-phosphorothioate polymers.

The term “small molecule” as used herein refers generally to a lowmolecular weight compound, usually less than 900 daltons.

The term “click chemistry” or “Copper-Catalyzed Azide-AlkyneCycloaddition reaction” or “CuACC reaction” as used herein, refers tothe copper(I)-catalyzed [3+2]-Huisgen 1,3-dipolar cyclo-addition ofterminal alkynes and azides leading to 1,2,3-triazoles. It may alsorefer to a copper free variant of this reaction that might also be used.(J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang,I. A. Miller, A. Lo, J. A. Codelli, C. R. Bertozzi, Proc. Natl. Acad.Sci. U.S.A. 2007, 104, 16793.).

As described herein, in accordance with the various embodiments herein,the inventors have developed a novel cell engineering technology bywhich molecules are covalently attached on the glycans present on a cellsurface. Such molecules may be small chemical moieties, or largerbiological moieties such as proteins, polynucleotides, and/orantibodies. The applications of the engineered cells disclosed hereinare numerous - illustrative examples include boosting the activity ofimmune cells for adoptive cell therapy (ACT); and/or conjugating smallmolecules, proteins or antibodies to the cell-surface for novelimmunotherapy strategies.

In one embodiment, disclosed herein is a non-naturally occurringengineered cell, comprising: a chemical or biological moiety covalentlybound to a glycan present on the surface of a cell via a fucosederivative. A variety of chemical and biological moieties can beattached to the cell surface glycan, as disclosed throughout thisdisclosure. Examples include, but are not limited to, drugs (e.g.,pharmaceuticals), catalysts, antibiotics, antibodies, antimycotics,carbohydrates, cytokines, enzymes, glycoproteins, lipids, nucleic acids,nucleotides, oligonucleotides, polynucleotides, proteins, peptides,ligand, cells, ribozymes, probe molecules, or combinations thereof.Similarly the chemical or biological moieties may be attached todifferent types of glycans present on the cell surface - non-limitingexamples include Gal (Galactin), GlcNAc (N-Acetylglucosamine), LacNAc,and/or NeuAc (Sialic acid). In one embodiment, this technique for makingengineered cells by covalently attaching molecules covalently on theglycans present on the cell surface is done in one pot, without the needfor purification. In another embodiment, this technique is done in onepot, one step, and in-situ.

In one aspect, the present disclosure provides an engineered cell,comprising a chemical or biological moiety covalently bound to a cellsurface glycan present on the surface of the cell, wherein the chemicalor biological moiety is selected from the group consisting of smallmolecule, polynucleotide, polypeptide, and antibody. In one embodiment,the engineered cell is an immune cell. In one embodiment, the engineeredcell is a T-cell, a Dendritic Cell (DC), or a natural killer (NK) cell.In one embodiment, the T cell is a CD8+ or a CD4+ T cell. In oneembodiment, the small molecule is a small drug molecule or apharmaceutically acceptable salt or a co-crystal thereof. In oneembodiment, the antibody is a single chain variable fragment (scFv),fragment antigen binding (Fab) fragment, or a full length antibody, suchas a full length immunoglobulin G (IgG) antibody. In one embodiment, thechemical or biological moiety attached to the engineered cell is abiological marker and/or probe. In one embodiment, the chemical orbiological moiety is a biotin probe, a fluorescent probe, a biorthogonalreaction handle, and/or a dye labeled single strand DNA. In oneembodiment, the dye is FAM. In one embodiment, the fluorescent probe isCy3. In one embodiment, the biorthogonal reaction handle is tetrazine.In one embodiment, the fucose derivative is a GDP-fucose. In oneembodiment, the engineered cell is a chimeric antigen receptor (CAR)-Tcell. In one embodiment, the CAR-T cell comprises a genetically modifiedT-cell with the cell surface GlcNAc covalently bound to a GDP-Fucosebearing a new motif. In one embodiment, the CAR comprises three domains:scFv, Fab, and/or mature ligands that engage their cognate receptor. Inone embodiment, the cell surface glycan is N-acetylglucosamine (GlcNAc).In one embodiment, the chemical or biological moiety is covalently boundto the GlcNAc via a fucose derivative. In one embodiment, the cellsurface glycan is Sialic acid (NeuAc).

In one embodiment, the present disclosure provides a compositioncomprising: an antibody-cell conjugate, wherein one or more antibodiesare covalently bound to one or more glycan moiety on the surface of acell. In one embodiment, the cell is immune cell. In one embodiment, thecell is a primary human T cell, a natural killer (NK) cell, a CD4+ cell,and/or primary CD8+ OT-1 T cells. In one embodiment, the NK cell isNK-92MI cell. In one embodiment, the cell is a Dendritic Cell (DC). Inone embodiment, the antibody is Trastuzumab. In one embodiment, the cellis NK-92MI and at least one antibody is Trastuzumab. In one embodiment,more than one type of antibody is conjugated on the surface of the cell.In one embodiment, the glycan moiety is N-acetylglucosamine (GlcNAc). Inone embodiment, the antibody is covalently bound to the GlcNAc via afucose derivative. In one embodiment, the glycan moiety is Sialic acid(NeuAc). In one embodiment, the composition is a pharmaceuticalcomposition. In one embodiment, the pharmaceutical composition furthercomprises a pharmaceutically acceptable carrier or excipient. In oneembodiment, the pharmaceutical composition is for the treatment of adisease in a subject. In one embodiment, the antibody-cell conjugateenables firm binding on inflammation sites (anti-E-selectin), targetspecific cancer cells (anti-HER2), and/or block the immune checkpoint(anti-PD-L1).

Cancer immunotherapy, which harnesses the body’s own immune system tofight cancer, has emerged as a fourth way of treating cancer, besidessurgery, radiation, and chemotherapy. While good results have beenachieved in adoptive cell therapy (ACT) and checkpoint blockade therapy,the cancer fighting properties of the T-cells infused in the patientsremain to be improved. Likewise, dendritic cells (DCs) have long beenconsidered as the central players in cancer immunotherapy. However, itwas shown that only a small portion (1-2%) of total administered DCsreached secondary lymphatic organs to activate T cells, limiting thepractical application of this vaccination approach.

In one embodiment, the engineered cells and methods disclosed hereinprovides an attractive way of overcoming these limitations. In oneembodiment, the present disclosure provides a method of treating adisease in a subject comprising: providing a pharmaceutical compositioncomprising an engineered cell and a pharmaceutically acceptable carrier;and treating the disease by administering a therapeutically effectivedosage of the pharmaceutical composition to the subject, wherein theengineered cell comprises a chemical or biological moiety covalentlybound to a glycan moiety present on its surface, and wherein thechemical or biological moiety is selected from the group consisting ofsmall molecule, polynucleotide, polypeptide, and antibody. In oneembodiment, the antibody is an immunoglobulin G (IgG) antibody. In oneembodiment, the antibody is an anti-PD-L1 antibody. In one embodiment,the antibody is an anti-E-selectin antibody. In one embodiment, theantibody is an anti-HER2 antibody. In one embodiment, the antibody isTrastuzumab. In one embodiment, more than one chemical or biologicalmoiety is covalently bound to the surface of the cell to target morethan one disease at the same time. In one embodiment, the cell is adendritic cell. In one embodiment, the cell is an immune cell. In oneembodiment, the cell is a primary human T cell, a natural killer (NK)cell, a CD4+ cell, and/or primary CD8+ OT-1 T cells. In one embodiment,the NK cell is NK-92MI cell. In one embodiment, the glycan moiety isN-acetylglucosamine (GlcNAc). In one embodiment, the chemical orbiological moiety is covalently bound to the GlcNAc via a fucosederivative. In one embodiment, the fucose derivative comprisesfucose-alkyne. In one embodiment, the glycan moiety is Sialic acid(NeuAc). In one embodiment, the disease is cancer. In one embodiment,treating the disease comprises reducing the size of a cancerous tumor inthe subject. In one embodiment, the cancer is a breast cancer.

In another embodiment, the present disclosure provides a method oftreating, decreasing, inhibiting, or reducing cancer in a subject,comprising: administering to the subject a therapeutically effectivedosage of a pharmaceutical composition comprising an engineered cell,having an antibody covalently bound to a glycan moiety present on itssurface. In one embodiment, the cancer is breast cancer. In oneembodiment, the antibody is Trastuzumab. In one embodiment, the cell isNK-92MI. In one embodiment, more than one antibody is conjugated on thesurface of the cell to target more than one cancer at the same time.

The present disclosure further provides a variety of methods for makingthe engineered cells disclosed herein. In one embodiment of the presentdisclosure, a method is provided for making an engineered cell,comprising: making a fucose derivative or GDP-fucose derivativecomprising a chemical or biological moiety covalently bound to a fucoseor GDP-fucose; making the engineered cell by incubating a cell with acomposition comprising (a) the fucose derivative and/or GDP-Fucosederivative and (b) a fucosyltransferase enzyme. In another embodiment ofthe present disclosure, a method is provided for making an engineeredcell, comprising: generating CMP-sialic acid conjugated with an antibody(CS-IgG) by reacting an antibody bearing a TCO moiety with CS-Az-Tz orCS-Poc-Tz; and making the engineered cell by incubating a naturallyoccurring cell with a composition comprising (a) CS-IgG and (b) asialyltransferase. In one embodiment, the fucosyltransferase enzyme isα-1,3-fucosyltransferase. In one embodiment, theα-1,3-fucosyltransferase is H. pylori α-1,3-fucosyltransferase. In oneembodiment, the α-1,3-fucosyltransferase is recombinantly prepared. Inone embodiment, the sialyltransferase is selected from the groupconsisting of ST6Gall, Pasteurella multocida α(2,3) sialyltransferaseM144D mutant (Pm2,3ST-M144D), and Photobacterium damsel α(2,6)sialyltransferase (Pd2,6ST). In one embodiment, the chemical orbiological moiety is selected from the group consisting of small drugmolecules, biomolecules, probe molecules, fluorophores, polynucleotides,polypeptides, and whole IgG, or combinations thereof. In one embodiment,the biomolecule is Trastuzumab. In one embodiment, the cell is an immunecell. In one embodiment, the cell is a primary human T cell or a naturalkiller (NK) cell. In one embodiment, the NK cell is NK-92MI cell. In oneembodiment, the T cell is a CD4+ cell, or a CD8+ T cells. In oneembodiment, the chemical or biological moiety is covalently attached viaa fucose derivative to LacNAc, a universal unit of N-glycans, on thesurface of the cell. In one embodiment, the fucose modified biomoleculeand/or GDP-fucose modified biomolecule comprises a fucose-alkyne and/orGDP-fucose azide. In one embodiment, the biomolecule-cell conjugate isfurther modified with a chemical or biological moiety using ligandaccelerated and biocompatible copper(I)-catalyzed alkyne-azidecycloaddition (CuAAC) reaction.

In one embodiment, disclosed herein is a one-pot method of making anengineered cell adapted to attach a molecule on its surface comprising:preparing GDP-fucose analog by combining a fucose analog with a mixturecomprising ATP, GTP, L-fucokinase/GDP-fucose pyrophosphorylase (FKP),Mg²⁺ or Mn²⁺, and inorganic pyrophosphate (PPase); and making theengineered cell by adding a naturally occurring cell to a compositioncomprising the crude product from step (a) and H. pyloriα-1,3-fucosyltransferase. In one embodiment, disclosed herein is aone-pot, one-step method of making an engineered cell adapted to attacha molecule on its surface comprising: incubating a cell with acomposition comprising a fucose analog, ATP, GTP,L-fucokinase/GDP-fucose pyrophosphorylase (FKP), Mg²⁺ or Mn²⁺, inorganicpyrophosphate (PPase) and H. pylori α-1,3-fucosyltransferase. In oneembodiment, the fucose analog comprises a fucose conjugated with achemical or biological moiety. In one embodiment, the fucose analogcomprises a fucose-alkyne. In one embodiment, the engineered cellcomprises a chemical or biological moiety attached to a glycan on thesurface of the cell. In one embodiment, the product from step (a) isfurther modified through a Copper-Catalyzed Azide-Alkyne Cycloaddition(CuACC) reaction to generate GDP-fucose analogs conjugated to a smallmolecule, polynucleotide, polypeptide, and/or antibody.

In one embodiment, disclosed herein is a one pot in-situ fucosylationstrategy to convert cell surface LacNAc or SLacNAc into LeX or sLeXcomprising: preparing GDP-fucose analogs by combining a fucose analogwith a mixture comprising ATP, GTP, FKP, Mg²⁺ or Mn²⁺, and inorganicpyrophosphate (PPase); and converting cell surface LacNAc or SLacNAcinto LeX or sLeX by adding a naturally occurring cell to a compositioncomprising the crude product from step (a) and H. pyloriα-1,3-fucosyltransferase.

In one embodiment, disclosed herein is a composition comprising anengineered cell is adapted to attach a molecule on its surface. In oneembodiment, the engineered cell is an immune cell. In one embodiment,the engineered cell is a T-cell. In one embodiment, the moleculeattached to the cell surface is a small molecule drug, an antibody, atherapeutic cell, a polypeptide and/or a polynucleotide. In oneembodiment, the antibody is a full length antibody. An antibody used inaccordance with this disclosure may be a polyclonal, monoclonal,chimeric, mouse, humanized or fully human. In one embodiment, themolecule attached to the engineered cell is a single-chain variablefragment (scFv). In one embodiment, the molecule attached to theengineered cell is a fragment antigen-binding (Fab) fragment. In oneembodiment, the molecule attached to the engineered cell is a biologicalmarker and/or probe. In one embodiment, the biological marker and/orprobe is a fluorescent dye. In one embodiment, the engineered cell is achimeric antigen receptor (CAR)-T cell wherein the CAR-T cell comprisesa genetically modified T-cell with the cell surface glycans covalentlybound to a GDP-Fucose bearing a new motif. In one embodiment, the CARmolecule comprises 3 domains: scFv, Fab, and/or mature ligands thatengage their cognate receptor.

The present disclosure describes a one-pot in situ fucosylation strategyto modify cell-surface glycans. The applications include (1) boost theactivity of immune cells for adoptive cell therapy (ACT); (2) conjugatesmall molecules, proteins or antibodies to the cell-surface for novelimmunotherapy strategies.

Almost all key cell-surface molecules involved in the innate andadaptive immune systems are glycosylated. Glycosylated molecules playessential roles in the immune cell differentiation and trafficking. Theinteraction between the glycan ligand sialyl Lewis X (sLe^(X)) with theselectin family of glycan binding proteins (P-selectin and E-selectin)mediates the tethering and rolling of circulating leukocytes on thevascular cell wall, which promotes subsequent extravasation andmigration of leukocytes through the endothelium into the surroundingtissue. However, the inventors have discovered that many activatedimmune cells with therapeutic potential such as lymphocytes or myeloidcells have very low levels of sLe^(X).

The inventors have solved this problem by disclosing herein a one-pot insitu fucosylation strategy to convert cell-surface N-acetyllactosamine(LacNAc, Galβ1,4 GlcNAc) or its sialylated form (sLacNAc) into the LewisX (Le^(x)) or sialyl Lewis X (sLe^(X)) epitopes. Converting cell-surfaceLacNAc and sLacNAc residues into sLe^(x) ex vivo on the surface ofimmune cells via this strategy serve as a “two birds, one stone”approach to boost the efficacy of cell-based immunotherapy. The sLe^(X)epitope created on the surface of immune cells directs the homing,engraftment and retention of therapeutic cells to diseased tissues wherethey are needed most. Furthermore, by converting LacNAc and sLacNAcresidues into sLe^(X), galectin-LacNAc interactions are inhibited, whichin turn suppresses galectin-mediated immune cell apoptosis. Using thisone-pot in situ fucosylation approach, biomacromolecules such as anantibody can be directly attached onto the cell surface of immune cellsfor immunotherapy.

Accordingly, in one embodiment, disclosed herein is a one-pot method ofmaking an engineered cell adapted to attach a molecule on its surfacecomprising: preparing GDP-fucose analog by combining a fucose analogwith a mixture comprising ATP, GTP, FKP, MgSO4, and inorganicpyrophosphate (PPase), and making the engineered cell by adding thecrude product from step (a) and α -1,3-FucT to a cell. In oneembodiment, disclosed herein is a one-pot, one-step method of making anengineered cell adapted to attach a molecule on its surface comprising:incubating a cell with a fucose analog, ATP, GTP, FKP, MgSO4, inorganicpyrophosphate (PPase) and α -1,3-FucT. In one embodiment, the engineeredcell comprises a molecule attached to a glycan on the surface of thecell. In one embodiment, the fucose analog comprises a fucose-alkyne. Inone embodiment, the product from step (a) is further modified through aCuACC reaction to generate GDP-fucose analogs conjugated to a smallmolecule, polynucleotide, polypeptide, and/or antibody. In oneembodiment, disclosed herein is a one pot in-situ fucosylation strategyto convert cell surface LacNAc or SLacNAc into Le^(X) or sLe^(X)comprising: Preparing GDP-fucose analogs by combining a fucose analogwith a mixture comprising ATP, GTP, FKP, MgSO4, and inorganicpyrophosphate (PPase); and Making the engineered cell by adding to acell a composition comprising the crude product from step (a) and H.pylori α-1,3-FucT. While the inventors have generally usedα-1,3-FucosylTransferase enzyme derived from the organism H. pylori, itwill be apparent to one of skill in the art that any fucosyltransferaseenzyme, irrespective of the source, may be used for the methods,compositions, and kits described herein.

In one embodiment, provided herein is a method of treating a disease ina subject comprising providing an engineered cell adapted to attach amolecule on its surface, and treating the disease by administering tothe subject the engineered cell. In one embodiment, provided herein is amethod of treating cancer in a subject comprising: providing anengineered cell wherein an anti-PD-L1 antibody is covalently linked tothe cell surface LacNAc molecule of an immune cell; administering to thesubject the engineered cell, wherein the antibody in the engineered cellrecognizes a specific antigen on cancer cells; and treating cancer inthe subject, by contacting the engineered cell with a cancer cell. Inone embodiment, the engineered cell comprises a molecule attached to aglycan on the surface of the cell. In one embodiment, the fucosecomprises fucose-alkyne. In one embodiment, the product from the firststep is further modified through a CuACC reaction to generate GDP-fucoseanalogs conjugated to a small molecule, polynucleotide, polypeptide,and/or antibody. In one embodiment, the size of the cancerous tumor inthe patient decreases upon administration of the engineered cell to thepatient.

Engineering the immune cell surface from the outside is an efficientapproach to improve cell-based therapy, which would have the potentialto compete with the genetic manipulation due to its low risk oflymphocyte transformation and genome mutation, or may be used as analternative approach, at least. Cell surface glycans are positioned mostoutside of the cell surface and therefore are excellent targets to beengineered. They could be efficiently modified through the metabolicengineering or chemoenzymatic engineering. As discussed throughout thisdisclosure, the inventors have developed a one-pot in-situ fucosylationstrategy to convert cell-surface LacNAc or its sialylated form sLacNAcinto the Le^(X) or sLe^(X) epitopes. This method has been widely used toboost cell therapy efficiency, such as stem cell, regulatory cell andcord blood cell based therapy. The inventors extended the one-potfucosylation strategy to install different functionalities, ranging fromsmall molecules to biomacromolecules, like whole IgG antibody, onto cellsurface. The process catalyzed by H. pylori α-1,3 fucosyltransferase isfast and efficient, even using a big substrate such as a proteinconjugated to GDP-fucose. Taken together, the inventors have developed atotally new method for engineering immune cells, which is biocompatibleand base on the natural epitope mimics, and also open the door ofplaying games with different combinations of small molecules drugs,antibodies and therapeutic cells.

For example, T cells can be genetically engineered to express specialreceptors on their surface called chimeric antigen receptors (CARs).CARs are proteins that allow the T cells to recognize a specific protein(antigen) on tumor cells. These engineered CAR T cells are then grown exvivo until they number in the billions. The expanded population of CAR Tcells is then infused into the patient. After the infusion, if all goesas planned, the T cells multiply in the patient’s body and, withguidance from their engineered receptor, recognize and destroy cancercells that harbor the antigen on their surfaces. A CAR may consist ofthree domains, i.e. antigen binding domain, transmembrane domain andcytosolic signal activation domain. The antigen binding domain falls in3 general categories: (a) single-chain variable fragment (scFv) derivedfrom antibodies; (b) Fab fragment antigen-binding (Fab) selected fromlibraries; or (c) nature ligands that engage their cognate receptor.CAR-Ts have showed promising efficacy in treating blood cancers,however, disappointing results were observed in the treatment of solidtumors due to the presence of suppressive mechanisms in the tumormicroenvironment.

The one-pot strategy can be applied to install small molecules, scFv,Fab or full-length antibodies targeting checkpoint inhibitors onto thesurface of CAR-Ts, ex vivo expanded tumor infiltrating lymphocytes orother types of immune cells to boost their activities. For example,using this strategy, the inventors can introduce antibodies blockingcheckpoint inhibitors, e.g. (Programmed Death ligand 1 PD-L1) and(Cytotoxic T Lymphocyte-Associated Antigen 4) CTAL-4, onto the surfaceof CAR-T cells (or ex vivo expanded tumor infiltrating lymphocytes).Similarly, the inventors can use the same strategy to introduceantibodies against CTAL-4 onto the surface of ex vivo maturated,antigen-loaded dendritic cells to boost their anti-tumor or antiviralactivities. Furthermore, using this strategy, the inventors can installtwo or more different molecules onto cell-surface, e.g. anti-PD-L1 andanti-Tim 3 to inhibit more than one checkpoint inhibitors. The samestrategy can be used to install biomolecules other than antibodies ontothe cell surface. For example, Programmed cell death protein 1 can beconjugated to regulatory T cells to enhance their immune suppressivefunctions.

Conjugating an antibody such as anti-PD-L1 to the cell surface of Tcells for combination immunotherapy can significantly reduce thequantity of the antibody to be administered. In clinic trials,anti-PD-L1 antibody is administered intravenously (at escalating dosesranging from 0.3 to 10 mg per kilogram of body weight) to patient every14 days in 6-week cycles for up to 16 cycles. By administratinganti-PD-L1 conjugated T cells, 10 to 100 fold less anti-PD-L1 would beused.

Thus, as disclosed herein, the inventors developed a novel method toengineer cells by construction of antibody-cell conjugates for enhancedcell therapy via one-step enzymatic glycoengineering. As describedherein, in accordance with the various embodiments herein, the inventorshave described a novel one-pot in situ fucosylation strategy to labelantigen specific antibody onto a natural killer (NK) cell surface, whichdirect NK cells to kill specific antigen expressing cancer cells. UsingHer2+ expressing cancer model, the inventors have demonstrated thatenzymatic conjugation of Herceptin to NK cells enhanced killing of Her2+expressing cancer cells in vitro and in vivo. The method is much easierand more efficient than genetic approaches.

In one embodiment, the present disclosure provides a compositioncomprising: an antibody-cell conjugate, wherein one or more antibodiesare covalently attached to one or more glycan molecules on the surfaceof the cell. In one embodiment, the cell is immune cell. In oneembodiment, the cell is a primary human T cell, a natural killer (NK)cell line, and/or primary CD8+ OT-1 T cells. In one embodiment, the NKcell line is NK-92MI. In one embodiment, the antibody-cell conjugate isas described in FIG. 1B. In one embodiment, the antibody is Trastuzumab(Herceptin). In one embodiment, more than one type of antibody isconjugated on the surface of the cell. In one embodiment, the cell isNK-92MI and at least one antibody is Trastuzumab (Herceptin). In oneembodiment, the antibody-cell conjugate enables firm binding oninflammation sites (anti-E-selectin), target specific cancer cells(anti-HER2), and/or block the immune checkpoint (anti-PD-L1).

In one embodiment, the instant disclosure provides a pharmaceuticalcomposition comprising: an antibody-cell conjugate as described above,and a pharmaceutically acceptable carrier or excipient. In oneembodiment, the pharmaceutical composition is for the treatment of adisease in a subject. In one embodiment, provided herein is a method oftreating, decreasing, inhibiting, or reducing cancer in a subject,comprising: administering to the subject a therapeutically effectivedosage of a pharmaceutical composition comprising an antibody-cellconjugate as disclosed herein. In one embodiment, the cancer is breastcancer. In one embodiment, more than one antibody is conjugated on thesurface of the cell to target more than one cancer at the same time.

In one embodiment, disclosed herein is a method of making abiomolecule-cell conjugate, comprising: incubating a cell with acomposition comprising (a) one or more fucose modified biomoleculesand/or GDP-Fucose modified biomolecules and (b) a fucosyltransferaseenzyme. In one embodiment, the fucosyltransferase enzyme isα-1,3-fucosyltransferase. In one embodiment, theα-1,3-fucosyltransferase is obtained from H. pylori. In one embodiment,the α-1,3-fucosyltransferase is recombinantly prepared. In oneembodiment, the biomolecules are probe molecules, fluorophores,polynucleotides, polypeptides, whole IgG, or combinations thereof. Inone embodiment, the biomolecule is Trastuzumab (Herceptin). In oneembodiment, the cell is an immune cell. In one embodiment, the cell is aprimary human T cell, a natural killer (NK) cell line, and/or primaryCD8+ OT-1 T cells. In one embodiment, the cell is NK-92MI. In oneembodiment, the biomolecule is covalently attached to LacNAc, auniversal unit of N-glycans, on the surface of the cell. In oneembodiment, the fucose modified biomolecule and/or GDP-fucose modifiedbiomolecule comprises a fucose-alkyne and/or GDP-fucose azide. In oneembodiment, the biomolecule-cell conjugate is further modified with achemical or biological moiety using ligand accelerated and biocompatiblecopper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. In oneembodiment, the chemical or biological moiety is a biotin probe, afluorescent probe Cy3, a biorthogonal reaction handle tetrazine, and/ora dye (FAM) labeled single strand DNA.

In one embodiment, using chemoenzymatic method to labeling antibodies toNK-92 cell surface provide a significant financial advantage and timesaving procedure. NK-92 cells do not express Fc receptors for ADCCeffects to target specific cells, which limit its wide applications.Genetic methods are used to install high affinity CD16 onto NK cellswhich can be used for in vivo treatment combined large dose of antibodyinjection. Using the chemoenzymatic labeling method, antibody conjugatedNK cells can be produced within hours and only consume low quantity ofantibody.

In one embodiment, the one-pot approach to transfer fucose linkedantibody to the cell surface provides a unique, specific andbiocompatible way to label two or more antibodies at the same time.Using the chemoenzymatic labeling system, two or more kinds of antigenspecific antibodies can be installed onto the same NK cell, whichfacilitated NK cells target different cancers at the same time, whilegenetic expression of two or more antibodies in the same cell is verydifficult.

In one embodiment, the composition disclosed herein is a pharmaceuticalcomposition. In various embodiments, the pharmaceutical compositionsaccording to the present disclosure may be formulated for delivery viaany route of administration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal or parenteral.“Parenteral” refers to a route of administration that is generallyassociated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or nonaqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject’s response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 21^(st) edition, Williams & Wilkins PA, USA) (2005).

Typical dosages of an effective composition can be in the rangesrecommended by the manufacturer where known therapeutic compounds areused, and also as indicated to the skilled artisan by the in vitroresponses or responses in animal models. Such dosages typically can bereduced by up to about one order of magnitude in concentration or amountwithout losing the relevant biological activity. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based, forexample, on the in vitro responsiveness of the relevant primary culturedcells or histocultured tissue sample, such as biopsied malignant tumors,or the responses observed in the appropriate animal models, aspreviously described.

In one embodiment disclosed herein is a kit comprising GDP-fucosederivative and H. pylori α-1,3-fucosyltransferase. The kit is useful forpracticing the inventive method of making an engineered cell adapted toattach a molecule on its surface. The kit is an assemblage of materialsor components, including at least one of the inventive compositions.Thus, in some embodiments the kit contains a composition including aGDP-fucose derivative and H. pylori α-1,3-fucosyltransferase, asdescribed above.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of treating cancer. In one embodiment, thekit is configured particularly for the purpose of treating mammaliansubjects. In another embodiment, the kit is configured particularly forthe purpose of treating human subjects. In further embodiments, the kitis configured for veterinary applications, treating subjects such as,but not limited to, farm animals, domestic animals, and laboratoryanimals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to make an engineered cell. Optionally, the kit also containsother useful components, such as, diluents, buffers, pharmaceuticallyacceptable carriers, syringes, catheters, applicators, pipetting ormeasuring tools, bandaging materials or other useful paraphernalia aswill be readily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging materials employed in the kit are those customarilyutilized in the chemical or biotechnological field. As used herein, theterm “package” refers to a suitable solid matrix or material such asglass, plastic, paper, foil, and the like, capable of holding theindividual kit components. Thus, for example, a package can be a glassvial used to contain suitable quantities of an inventive compositioncontaining GDP-fucose derivative and H. pylori α-1,3-fucosyltransferase.The packaging material generally has an external label which indicatesthe contents and/or purpose of the kit and/or its components.

Embodiments of the present disclosure are further described in thefollowing examples. The examples are merely illustrative and do not inany way limit the scope of the invention as claimed.

EXAMPLES Example 1

Cell engineers are continuously working with great effort on trainingcells to be living therapeutics. The past several years have witnessedspectacular results in patients treated with adoptive cell transfer(ACT). Most notably, Kymriah® (tisagenlecleucel) (a Registered trademarkof Novartis), a chimeric antigen receptor T-cell (CAR-T) therapy, isapproved as the first cell-based gene therapy in the United States mostrecently. Genetic engineering serves as a common and robust approach toengineer cells with novel functionalities. However, genetic engineeringhas showed limited success due to several technical challenges andsafety concerns (e.g. viral transduction resistance of primary cells,heterogeneous expression levels and potential of endogenous genedisruption). To break through these limitations, engineering cellsurfaces from “outside” via biochemical, biophysical or enzymaticmethods has emerged as versatile and general applicable approaches. Themodification sites of non-genetic methods from “outside” could beglycans, proteins or lipids on cell surface, among which glycans arepositioned at the interface between cells and the extracellular milieuand therefore serve as excellent targets. In cell surfaceglycoengineering, a two-step method has been developed for couplingmetabolic oligosaccharide engineering (MOE) with bioorthogonalchemistry. The advantages of high efficiency and good biocompatibilitymake it one of the most widely used cell surface engineering approaches.

Preparation of therapeutic cells in clinical practice usually needs exvivo isolation, activation and large-scale expansion of specific cells.Both genetic and metabolic engineering start in the early stages ofcells preparation (e.g. during activation) and might interfere withthese procedures. Moreover, these approaches are often completed throughmulti-steps in several days, sometimes followed by subsequent chemicalreactions, which need more cell processing and quality control outsideof the body (FIG. 39A). Their complexity in procedures could slow downthe clinical translation. In contrast, enzymatic engineering of cellsurface glycans as disclosed herein could be easily achieved in one-stepjust before infusion to human body. One impressive example of enzymaticengineering is ex vivo fucosylation of cells (e.g. stem cells andregulatory T cells) with GDP-fucose (GDP-Fuc or GF) and exogenousα(1,3)-fucosyltransferase (FucT or FT) could promote or improve theprocess of adhesion, homing or engraftment in adoptive transfer, whichis currently under several clinical trials. However, transferringnatural donor substrates to cell surface in glycoengineering has anarrow range of applications. To enrich the applications of cell surfaceenzymatic glycoengineering, expanding substrates scope to installversatile functionalities is highly desired.

Previously Palcic and co-workers described a method of decoratingerythrocytes of blood type O with a blood group B trisaccharide antigen,by taking advantage of the relaxed substrates specificity of a humanα1,3FucT that could recognize GDP-Fuc derivatives. (See Srivastava, G.;Kaur, K. J.; Hindsgaul, O.; Palcic, M. M. J. Biol. Chem. 1992, 267,22356.) While this protocol provided an important development - mostnotably enzymatic transfer of diversified unnatural functional moleculesto cell surface; several key drawbacks remained, which made the methodnot useful for therapeutic applications.

The presently disclosed in-situ fucosylation or sialylation strategyprovides an attractive alternative. The reaction system disclosed hereinis more efficient than previously disclosed methods, it is morebiocompatible, and can be used to transfer large molecules, such asproteins, polypeptides, polynucleotides, and antibodies to a cell.

In one embodiment, the present disclosure provides sialyltransferasesand fucosyltransferases that efficiently and site-specifically installsfunctionalities like bioorthogonal handles, fluorescent probes orpreassembled oligosaccharide on live cell surface for glycan detection,glycoprotein analysis or glycan architectures remodeling. Although novelapplications have appeared, the moieties that could be enzymaticallytransferred are still chemical synthesized small molecules or oligomers.Further, as disclosed herein, complex and biologically importantmacromolecules can be installed on a cell surface, and the resultingengineered cell would be broadly applicable in cell therapy. Forexample, monoclonal antibodies (mAbs) have shown great efficacy intreating disease like cancer or autoimmune. Making antibody-drugconjugates (ADC) would further enhance the potency of mAbs in cancertherapy. In one embodiment, enzymatic transfer of mAbs to therapeuticcell surface would transiently alter the functions of cells throughremodeling cell-cell interactions, blocking signaling pathways orgenerating synergetic therapeutic effects.

In one embodiment, disclosed herein is a novel strategy for constructingantibody-cell conjugates (ACC) using α1,3FucT from H. pylori, whichcould transfer a whole IgG antibody modified with GDP-Fuc derivatives toLacNAc (Galβ1,4GlcNAc), a universal unit of N-glycans, on live cellsurface in several minutes. Meanwhile, a one-pot protocol couplingchemoenzymatic reactions with bioorthogonal chemistry was also inventedto produce GDP-Fuc derivatives modified biomaterials from easilysynthesized fucose analogues, which makes this engineering approach moregeneral applicable and cost effective. In one embodiment, using thismethodology, three cases of ACC on immune cells were established,including primary human T cells, natural killer cell lines (NK-92MI) andprimary CD8+ OT-1 T cells. Monoclonal antibodies conjugated on cellsurfaces help these immune cells to enhance firm binding on inflammationsites (anti-E-selectin), target specific cancer cells (anti-HER2) orblock the immune checkpoint (anti-PD-L1), which might improve adoptivecell therapies in different stages.

Example 2

FIG. 1 illustrates one embodiment of the two step one-pot reaction, andthe one step one-pot reaction.

Two step one-pot reactions: Reactions were typically carried out in a1.5-mL Eppendorf tube with 1.0 mL buffer (pH 7.5) containing L-fucose orits C-5 substituted analogs (final concentration, 10 mM), ATP (10 mM),GTP (10 mM), MgSO4(10 mM), inorganic pyrophosphatase (90 units,endotoxin free), and FKP (9 units, endotoxin free). The reaction mixturewas incubated at 37° C. for 5-6 h with shaking (225 rpm). The reactionprogress was monitored by TLC, using 10 mM tetrabutylammonium hydroxidein 80% aqueous acetonitrile as the developing solvent (p-anisaldehydesugar stain). After the disappearance of fucose or fucose analogs, thecrude product could be used directly on cell-surface fucosylation. Iffucose-alkyne or azide were used, the crude product could be furthermodified through CuAAC reaction to generate GDP-fucose conjugates. Forexample, the crude GDP-fucose-alkyne sample (5 mM) were reacted withazide probes (5 mM) in the presence of Cu/BTTAA (100 uM) and sodiumascorbate (2 mM) at 37° C. for 1 h. After reaction finished, BCS (1 mM)were added to quench the reaction. The crude one-pot product, eitherbefore or after click reaction, could be directly used for thesubsequent fucosylation reaction on the cell surface withoutpurification. 10⁶ live cells were washed 3 × times with PBS andre-suspended in 100 µL HBSS buffer containing 20 mM MgSO4, 3 mM HEPES,0.1% FBS, 200 µM GDP-Fuc (or GDP-Fuc-conjugates), 30 mU α(1,3)FucT.After the incubation at 37° C. for 20 min, the cells were washed 3×times and then blocked in PBS with 2% FBS for 30 min for furtherapplication or analysis.

One-step one-pot reaction: Reagents were prepared in a 1.5-mL Eppendorftube with 100 uL buffer (pH 7.8) containing L-fucose or its C-5substituted analogs (final concentration, 1 mM), ATP (10 mM), GTP (10mM), MgSO4(20 mM), inorganic pyrophosphatase (90 units, endotoxin free),FKP (9 units, endotoxin free) and 30 mU H. pylori α(1,3)FucT (endotoxinfree). Live cells (1×10⁶) were then added to the reaction mixtures.After the incubation at 37° C. for 60 min, the cells were washed threetimes and then blocked in PBS with 2% FBS for 30 min for furtherapplication or analysis.

Example 3

As illustrated in FIG. 2 , in one embodiment, one-pot fucosylationreaction was performed on cultured CHO cells. After fucosylation, thecell surface sLacNAc were converted into sLe^(X), which could bedetected through APC conjugated anti-CLA antibody (FIG. 2A). Live CHOcells were washed 3 times with PBS and resuspended in 100 µLfucosylation buffer (containing H. pylori α-1,3-FucT) with eitherpurified GDP-fucose (indicates as normal reaction in the figure) orone-pot GDP-fucose. After the incubation at 37° C. for 20 min, the cellswere washed, blocked and then stained with APC-anti-CLA. After washing,the samples were analyzed in flow cytometry. As shown in FIGS. 2B and2C, the fucosylation reaction runs well with one-pot GDP-fucose. Theconcentration titration indicates that the reaction could be saturatedusing 100 uM one-pot GDP-fucose, which is identical to purifiedGDP-fucose according to the results disclosed herein. Controlexperiments were also characterized without H. pylori α-1,3-FucT orGDP-fucose (FIG. 2B), which indicates that the reaction needs both ofthe reagents. The control antibody staining was shown in FIG. 2D. (FIG.2C, One-way ANOVA test, between columns, P<0.0001)

Example 4

As illustrated in FIG. 3 , in one embodiment, one-pot fucosylationreaction was used to modify Lec2 CHO cells (with high level of LacNAcexpression) within biotin probe. The GDP-fucose-biotin derivative wassynthesized in one-pot according to the procedure in Example 1. One-potfucosylation reaction was used to modify Lec2 CHO cells (with high levelof LacNAc expression) with biotin probe. Using GDP-fucose-biotin as thesubstrate, the cell surface was fucosylated with biotin molecule, whichis specifically located on LacNAc (FIG. 3A). Live lec2 cells were washed3 times with PBS and resuspended in 100 µL fucosyaltion buffer(containing H. pylori α-1,3-FucT) with one-pot GDP-fucose-biotin. Afterthe incubation at 37° C. for 20 min, the cells were washed, blocked andthen stained with APC-streptavidin (APC-SA). After washing, the sampleswere analyzed in flow cytometry. As shown in FIGS. 3B and 3C, the lec2cells were efficiently labeled with biotin after fucosylation reactionusing the one-pot synthesized GDP-fucose-biotin. The concentrationtitration indicates that saturation concentration is reached when using50 uM one-pot GDP-fucose-biotin. Control experiments were alsocharacterized without H. pylori α-1,3-FucT or GDP-fucose-biotin (FIGS.3B, 3 and 4 ), which indicates that the reaction needs both of thereagents. The control antibody staining was shown in FIG. 3D. OtherGDP-fucose derivatives could also been synthesized and used to modifycells using the same protocol. This is a general approach to modify cellsurface specifically on LacNAc. (FIG. 3C, One-way ANOVA test, betweencolumns, P<0.0001) The biotinylated cells could be furtherfunctionalized with SA-conjugated moieties.

Example 5

As illustrated in FIGS. 4-8 , cell surfaces may be modified using theone-pot fucosylation reaction strategy disclosed herein.

In one embodiment, FIG. 4 illustrates modification of cultured Lec2 CHOcell surface using crude one-pot GDP-fucose-Cy3. In one embodiment, theone-pot fucosylation reaction was used to modify Lec2 CHO cells withfluorescent dye Cy3. GDP-fucose-Cy3 derivatives were synthesized by theone-pot procedure disclosed herein. Using GDP-fucose-Cy3 as substrates,the cell surface was modified with Cy3 probe specifically on LacNAc,which could be directly detected (FIG. 4A). After fucosylation, thesamples were analyzed in flow cytometry. As shown in FIGS. 4B and 4C,the Lec2 cells were efficiently labeled with Cy3 probe. Controlexperiments were also characterized without H. pylori α-1,3-FucT orGDP-fucose-Cy3 (FIG. 4B), which indicates that the reaction needs bothof the reagents. The Cy3 labled LacNAc on cell surface could be in situtracked using confocal microscope.

Example 6

In one embodiment, FIG. 5 illustrates modification of cultured Lec2 CHOcell surface using crude one-pot GDP-fucose-tetrazine. One-potfucosylation reaction was used to modify CHO-lec 2 cells with theefficient bioorthogonal group, tetrazine (Tz). GDP-fucose-tetrazinederivatives were synthesized by the one-pot procedure according to theprocedure in Example 1. Using GDP-fucose-Tz as substrates, the cellsurface was functionalized with tetrazine group specifically on LacNAc,which could be further efficiently conjugated with trans-cyclooctene(TCO) through the inverse-electron demand Diels-Alder reaction (inv DA)(FIG. 5A). The fucosylation protocol is the same as in Example 3. Afterfucosylation, the samples were conjugated with TCO-Cy5 probe for thedetection of tetrazine group and then analyzed in flow cytometry. Asshown in FIGS. 5B and 5C, the lec2 cells were efficiently labeled withCy5 probe. Control experiments were also characterized without 1, 3 FucTor GDP-fucose-Tz (FIG. 5B), which indicates that the reaction needs bothof the reagents.

Example 7

In one embodiment, FIG. 6 illustrates modification of cultured Lec2 CHOcell surface using crude one-pot GDP-fucose-BSA. One-pot fucosylationreaction was used to modify CHO-lec 2 cells with model BSA protein. BSAprotein was first reacted with the NHS-biotin and NHS-azide probe. Theazide groups on the BSA protein were then reacted with GDP-fucose-alkyneprobes through CuAAC reaction. After reaction, the mixture was quenchedwith BCS and then desalted. Using GDP-fucose-BSA as substrates, the cellsurface was conjugated with BSA protein specifically on LacNAc (FIG.6A). GDP-fucose-BSA concentration was 0.4 mg/ml in this example. Afterfucosylation, the samples were stained with APC-SA to detect the biotinprobe on BSA protein and then analyzed in flow cytometry. As shown inFIGS. 6B and 6C, the lec2 cells were efficiently labeled with BSAprotein. Control experiments were also characterized without H. pyloriα-1,3-FucT or GDP-fucose-BSA (FIG. 6B), which indicates that thereaction needs both of the reagents.

Example 8

In one embodiment, FIG. 7 illustrates modification of cultured Lec 2 CHOcell surface using crude one-pot GDP-fucose-antibody. One-potfucosylation reaction was used to modify Lec 2 CHO cells withantibodies. The rat IgG was first reacted with the NHS-azide probe. Theazide groups on the rat IgG were then reacted with GDP-fucose-alkyneprobes through CuAAC reaction. After reaction, the mixture was quenchedwith BCS and then desalted. Using GDP-fucose-IgG as substrates, the cellsurface was conjugated with rat IgG specifically on LacNAc (FIG. 7A).GDP-fucose-IgG concentration is 0.2 mg/ml in this example. Afterfucosylation, the samples were stained with APC-anti-Rat IgG to detectthe rat IgG and then analyzed in flow cytometry. As shown in FIGS. 7Band 7C, the lec2 cells were efficiently labeled with rat-IgG. Controlexperiments were also characterized without H. pylori α-1,3-FucT (FIG.6B).

Example 9

In one embodiment, FIG. 8 illustrates modification of cultured Lec2 CHOcell surface using crude one-pot GDP-fucose-Tz and subsequentTCO-antibody. As described above in Example 5, the CHO cell surface wasfunctionalized with tetrazine group specifically on LacNAc. Afterfucosylation, the samples were reacted with 0.1 mg/ml TCO-mouse IgG for20 min in room temperature (FIG. 8A). The TCO-IgG protein was firstprepared by label IgG with NHS-TCO group. After conjugated with mouseIgG antibody, the cells were stained with APC-anti-mouse IgG and thenanalyzed by FACS. As shown in FIGS. 8B and 8C, the lec2 cells wereefficiently labeled with mouse IgG. Control experiments were alsocharacterized without H. pylori α-1,3-FucT or GDP-fucose-Tz (FIG. 5B),which indicated that the reaction needs both of the reagents. Thisexample shows that the cells functionalized with tetrazine can be easilymodified by TCO-moieties.

Example 10

As illustrated in FIGS. 9-14 , cell surfaces may be modified using theone-pot fucosylation reaction strategy disclosed herein.

In one embodiment, FIG. 9 illustrates modification of cultured wild typeCHO cells surface using crude one-pot GDP-fucose-biotin. To illustratethe general applicability, one-pot fucosylation reaction was used tomodify wild type CHO cells with biotin probe using the protocoldisclosed in Example 3. After fucosylation and staining with SA-APC, thesamples were analyzed in flow cytometry. As shown in FIG. 9 , the CHOcells were efficiently labeled with biotin.

Example 11

In one embodiment, FIG. 10 illustrates modification of activated mouseCD8+ cells surface using crude one-pot GDP-fucose-biotin. To furtherillustrate the general applicability, one-pot fucosylation reaction wasused to modify activated mouse CD8+ T cells with biotin probe. Thephenotype of the cells was characterized first (FIG. 10 ). Most of thecells are CD3+CD8+ cells. The CD44 high expression indicates theactivation of the T cells. The cells were fucosylated and stained withSA-APC, and subsequently the samples were analyzed in flow cytometry. Asshown in FIG. 10 , the mouse CD8+ T cells were efficiently labeled withbiotin.

Example 12

In one embodiment, FIG. 11 illustrates modification of mouse dendriticcell (DC) surface using crude one-pot GDP-fucose-biotin. To furtherillustrate the general applicability, one-pot fucosylation reaction wasused to modify mouse DC cells with biotin probe. The phenotype of thecells was characterized first (FIG. 11 ). Most of the cells are CD11c+cells. After fucosylation and staining with SA-APC, the samples wereanalyzed in flow cytometry. As shown in FIG. 11 , the DC cells wereefficiently labeled with biotin.

Example 13

In one embodiment, FIG. 12 illustrates modification of naïve oractivated human CD8+ T cell surface using crude one-potGDP-fucose-biotin. To further illustrate the general applicability,one-pot fucosylation reaction was used to modify human CD8+ T cells withbiotin probe. The phenotype of the cells was characterized first (FIG.12 ). Most of the cells were CD3+CD8+ cells. The CD45RO high expressionindicates the activation of the T cells. After fucosylation and stainingwith SA-APC, the samples were analyzed in flow cytometry. As shown inFIG. 12 , both the naïve and activated human CD 8+ T cells were labeledwith biotin. The activated human CD8+ T cells have higher LacNAc leveland were labeled more efficiently.

Example 14

FIG. 13 illustrates one embodiment of the modification of activatedmouse CD8+ T cell surface using crude one-pot GDP-fucose-Tz and thenconjugated with TCO-mouse IgG. To illustrate the general applicability,one-pot fucosylation reaction was used to install IgG onto activatedmouse CD8+ cells surface. The phenotype of the cells was characterized,followed by fucosylation. After fucosylation with GDP-fucose-Tz, thecells were then conjugated with TCO-mouse IgG. After that, the cellswere staining with APC-anti-mouse IgG and then analyzed in flowcytometry. As shown in FIG. 13 , the CD 8+ T cells were efficientlylabeled with mouse IgG. Control experiments were also characterizedwithout α-1,3 FucT (FIG. 13 ), which indicates that the labeling isbased on unnatural fucosylation. The TCO-IgG does not have non-specificbinding (FIG. 13 ).

Example 15

FIG. 14 illustrates one embodiment of the modification of activatedmouse CD8+ T cell surface using crude one-pot GDP-fucose-Tz and thenconjugated with TCO-mouse IgG and TCO-rat IgG together. Mouse T cellsurface was functionalized with tetrazine group specifically on LacNAc.After fucosylation, the samples were reacted with 0.1 mg/ml TCO-mouseIgG and 0.1 mg/ml TCO-rat IgG together for 20 min in room temperature(FIG. 14 ). The TCO-IgG proteins were first prepared by label IgG withNHS-TCO group. After conjugated with IgG antibodies, the cells werestained with APC-anti-mouse IgG, FITC-anti-rat IgG and then analyzed byFACS. As shown in FIG. 14 , the mouse CD8+ T cells were efficientlylabeled with both mouse IgG and rat IgG. Control experiments were alsocharacterized with only TCO-Abs (Rat IgG has some non-specific bindingtowards mouse T cells) (FIG. 14 ). This example shows that the cellsfunctionalized with tetrazine can be easily modified with two differentIgG antibodies bearing TCO group.

Example 16

FIG. 15 illustrates one embodiment of installation of anti-PD-L1antibody on OT-1 T cell surface. As described above in Examples 8 and13, using GDP-fucose-Tz as substrate, cell surface may be modified withtetrazine group and then antibody is installed onto cell surface via invDA reaction between Tz and TCO group. In this example, anti-PD-L1antibody, which inhibits the immune check-point (PD-1/PD-L1 pathway),was covalently linked to cell surface LacNAc and sLacNAc via thisapproach. The OT-1 CD8+ T cells (sample 1) were fucosylated withGDP-fucose-Tz (sample 3) and then were incubated with anti-PD-L1antibody to block the PD-L1 protein on T cell surface (sample 4). Afterthat, the cells were stained with FITC-anti-rat IgG to mark the antibodythat bind to native expressed PD-L1. Later, the cells were conjugatedwith TCO-anti-PD-L1 antibody through inv DA reaction (sample 5). Atlast, the cells were stained with APC-anti rat IgG to show thecovalently linked anti-PD-L1 on cell surface. Control experiments wereperformed: sample 4 was mixed with anti-PD-L1, or sample 1 was directlyblocked with PD-L1 (sample 2) and then mixed with TCO-anti-PD-L1. Asshown in FIG. 15 , only the Tz bearing cells were covalently conjugatedwith anti-PD-L1 (APC channel, control samples have a little backgroundcompared to the blank cells). And all the control samples only showpositive signal in FITC channel, which indicates the binding ofanti-PD-L1 with native expressed PD-L1 protein. This example shows thatthis method could be used to modify T cells surface with functionalantibody.

Example 17

FIG. 16 illustrates one embodiment of installation of anti-PD-L1antibody on OT-1 CD8⁺ T cell surface to boost the adoptive cell therapy(ACT). As shown in Example 14, anti-PD-L1 antibody could be covalentlylinked to the OT-1 cell surface. In this example, the OT-1 CD8+ T cellswere also modified with anti-PD-L1 antibody after the native expressedPD-L1 protein was blocked (in this example, the samples were not stainedafter the PD-L1 antibody treatment). Murine B16-OVA melanoma cells(5×10⁵) were subcutaneously inoculated to the shaved flank of 8-week oldC57BL/6 female WT mice. After 10 days, 10⁵ of OT-1 CD8+ T cells (withdifferent modification, sample 1, 4 and 5) were adoptively transferredto those B16-OVA tumor bearing mice (day 0, n=2 in each group).Subsequently, 10⁵ (on day 5) of the same processed OT-1 CD8+ T cellswere transferred in each experimental mouse. Tumor sizes were determinedone or two days by caliper measurements. At day 11, one of the mice insample 1 group died and all the other mice were euthanized and the tumorof each mouse was weighed (FIG. 16 ). The tumor mass and growth curveboth indicate that adoptive transfer of T cells with covalently linkedanti-PD-L1 antibody have significant improvement in the cancer therapy.

Example 18

FIG. 17 illustrates one embodiment of in-situ one-pot fucosylation of invivo expanded mouse CD8+ T cells increase E-selectin binding andsuppress Gal-1 binding. As shown in FIG. 17A, spleenocytes fromOT-1+/-CD45.1+/- mice were collected and sorted to get pure CD8+ naïve Tcells. 10⁵ of these fresh T cells were injected into wild type B6 miceretroobitally. Half day later, 10⁴ lysteria-ova cells were alsoretroobitally injected to these mice to activate those naïve OT-1 CD8+ Tcells. After several days of expansion, CD8+T cells from WT recipientmice were sorted and then fucosylated through one-pot reaction. Afterfucosyaltion, the cells were stained with E-selectin or Gal-1 protein,and then washed, stained with second antibody. After the stainingprocess, the samples were analyzed by flow cytometry. CD45.1 congenicmarker was used to track the OVA-antigen activated CD8+ T cells. Asshown in FIG. 17B, the E-selectin binding on OVA-specific CD8+ T cellsincreases significantly after fucosylation in each time point. In day14, the difference of E-selectin binding between fucosylated andunfucosylated is up to 10 fold. By contrast, the Gal-1 binding decreasesafter fucosylation (FIG. 17C).

Example 19

FIG. 18 illustrates one embodiment of in-situ one-pot fucosylation ofinduced mouse regulatory T cells (iTreg) increase E-selectin binding.Naive CD4+ T cells were sorted from SAMP or AKR mice spleenocytes. Thesecells were then re-suspended in T cell culture media and induced in apre-coated plate supplemented with 2 ug/ml a-CD28, 10 ng/ml IL 2, 20ng/ml TGF-b, 10 nM RA. The concentration of cells were kept at 1^10⁶/mlby adding media with IL2 and TGF-b. After 5 days expansion, more than80% of the viable cells were CD4+ CD25+ T cells as determined by flowcytometry. The ratio of FOXP3+ cells in CD4+CD25+ was higher than 90%,which indicate that most of these cells are iTreg cells (FIG. 18A). Onday 6 iTreg cells were then treated with one-pot fucosylation. Afterfucosylation, the E-selectin binding level was obviously improved (FIG.18B).

Example 20

FIG. 19 illustrates one embodiment of in-situ one-pot fucosylation ofhuman T cells that results in increased E-selectin binding. Human PBMCswere isolated by Ficoll density gradient centrifugation from human bloodand then activated with plate bound anti-CD3 (5 ug/ml) and solubleanti-CD28 (2 ug/ml). Twenty-four hours after activation, media werechanged and PBMCs were cultured in fresh T cell media with IL 2 andexpanded for approximately 2 weeks (kept at 1^10⁶ cells/ml). After 14days of expansion, more than 95% of the total cultured PBMCs were CD45 +CD3+ T cells, determined by flow cytometry, and about 60% of T cellsexpressed the CD8+ phenotype. The expanded human T cells were thanfucosylated with one-pot fucosylation reagents. After fucosylation, theE-selectin binding level was increased about 8-fold (orange vs. blueFIG. 19 ).

Example 21

FIG. 20 illustrates one embodiment of in-situ one-pot fucosylation ofhuman natural regulatory T (nTreg) cells increase E-selectin binding andinhibit Gal-1 binding. Human PBMCs were isolated by Ficoll densitygradient centrifugation from human blood. Human nTreg cells are sortedas CD4+CD127lowCD25+ cells. After sorting, nTreg cells were kept at1^10⁶ cells/ml in T cell culture media with IL2. After 14 daysexpansion, ~90% cells are FOXP3+ cells (FIG. 20A). After one-potfucosyaltion, the E-selectin binding was increased and Gal-1 binding wasslightly decreased (FIGS. 20B and 20C).

Example 22

FIG. 21 illustrates one embodiment of in-situ one-pot fucosylation ofHer2 specific human CAR-T cells increases E-selectin binding andinhibits Gal-1binding. Human PBMCs were isolated by Ficoll densitygradient centrifugation from human blood and then activated with platebound anti-CD3 (5 ug/ml) and soluble anti-CD28 (2 ug/ml). Twenty-fourhours after activation, PBMCs were transduced with lentiviral vectors ata multiplicity of infection of 5 and then cultured in fresh T cell mediawith IL 2 and expanded for approximately 2 weeks (kept at 1^10⁶cells/ml). After 14 days of expansion, more than 95% of the totalcultured PBMCs were CD45 + CD3+ T cells, determined by flow cytometry,and about 90% of T cells expressed Her2 specific CAR-T cells (FIG. 21A).These Her2 specific CAR T cells were then seeding into cultures of Her2+SKBR3 cells or Her2- MDA-MB-468 cells. After 24 hours of T-cell seeding,formation of T-cell clusters and elimination of the cancer cellmonolayer were only visible in Her2+ SKBR3 cells (FIGS. 21B, 21C). Theseresults indicate that Her2-specific T cells recognize and kill Her2+cells in a Her2-specific manner. The expanded human CAR-T cells werethen fucosylated with one-pot fucosylation reagents. After fucosylation,the E-selectin binding level was increased about 4 fold (FIG. 21D),while the Gal-1binding was only decreased slightly (FIG. 21E).

Example 23

FIG. 22 illustrates one embodiment of in situ one-pot fucosylation of invitro differentiated mouse CD8 T cells significantly enhanced E-selectinbinding and decreased Gal-1 binding. (A) Splenocytes of OT-1+/+Rag-/-mice were incubated with 1 nM SIINFEKL peptide in growth media. Threedays later, the cells were cultured in RPMI medium supplemented withIL-15 20 ng/ml and IL-7 10 ng/ml. Media were replaced and cytokines wereadded every 2 days. After 3 days of cytokine treatment, more than 95% ofthe viable cells were CD8+ T cells as determined by flow cytometry.Equal numbers of the CD8+ T cells were incubated in in situ fucosylationreaction buffer with fucose, GTP, ATP, FKP and H. pyloriα-1,3-fucosyltransferase (one-pot reaction) or GTP, ATP, FKP and H.pylori α-1,3 fucosyltransferase without fucose (control) at 37° C. for30 min. (B) Both the in situ fucosylated and non-fucosylated (ascontrol) cells were stained with biotinylated Galectin-1 and humanFc-conjugated E-selectin, respectively, washed, then stained withstreptavidin-PE and anti-human-Fc-PE, respectively, and analyzed usingflow cytometry.

Example 24

FIG. 23 illustrates one embodiment of in situ one-pot fucosylation of invitro differentiated mouse OT-1 CD8 T cells had little impact on theexpression of cell-surface markers. Splenocytes of OT-1+/+Rag-/- micewere incubated with 1 nM SIINFEKL peptide in growth media. Two dayslater, the cells were cultured in RPMI medium supplemented with IL-2 orIL-15 or IL-15+IL-7. After 3 days of cytokine treatment, more than 95%of the viable cells were CD8+ T cells as determined by flow cytometry.Equal numbers of the CD8+ T cells were incubated in in situ fucosylationreaction buffer with fucose, GTP, ATP, FKP and α 1,3 fucosyltransferase(one-pot reaction) at 37° C. for 30 min or untreated. Cell-surfacemarkers were stained with corresponding antibodies and analyzed usingflow cytometry.

Example 25

FIG. 24 illustrates one embodiment of treatment of B16-OVA-embeded micewith in vitro differentiated, glycan modified OT-1 T cells significantlydecreased tumor growth. Murine B16-OVA melanoma cells (6×10⁵) weresubcutaneously inoculated to the shaved flank of 8-week old C57BL/6female WT mice. After 8 days, 10⁵ of in situ fucosylated ornon-fucosylated IL-15+IL-7 in vitro differentiated OT-1 CD8+ T cellswere adoptively transferred to those B16-OVA tumor bearing mice (day 0,n=10 in each group). Subsequently, 2×10⁵ (on day 6) and 5×10⁵ (on day 9)of the same processed OT-1 CD8+ T cells were transferred in eachexperimental mouse. Tumor sizes were determined daily by calipermeasurements. * P<0.05 (determined by non-parametric t-test).

Example 26

FIG. 25 illustrates one embodiment of treatment of B16-OVA- embeddedmice with in vivo differentiated, glycan modified OT-1 T cellssignificantly decreased tumor growth and extended the life span of thetumor embedded mice. (A) To obtain OT-1 memory CD8+T cells in vivo,1×10⁴ OT-1 naïve CD8 T cells were transferred to CD45.1 +/+ micefollowed by infection with 1×10⁴ CFU of Listeria monocytogenes strainexpressing OVA (Lm-OVA) after 3 hr. After 7 days, the recipient micewere infection with 1×10⁵ vesicular stomatitis virus encoding ovalbumin(VSV-OVA) to boost OVA-specific immune responses. 21 days later, CD8 Tcells containing about 3-5% OVA-specific OT-1 memory CD8 T cells can beharvested from CD45.1 mice splenocytes by negative selection. After insitu fucosylation or untreated, the CD8 T cells containing 1×10⁵ OT1memory CD8 T cells were i.v. injected to WT mice bearing B16-OVA tumoron their flanks, which were s.c. implanted at 5×10⁵ for each mouse 10days ago. The memory T cells were injected three times on day 0, day 5and day 10. (B) OT-1 memory CD8+T cell treated with in situ fucosylationor untreated as described above were adoptively transferred in to micebearing B16-OVA tumors (n=5 in each group). Tumor size was monitored bycaliper measurement every other day and mice were sacrificed when tumorsize reached 1.8 cm. Data (mean+/- s.e.m.) are representative of threeindependent experiments. * p<0.05, **p<0.005 (analyzed by non-parametrict-test). (C) Mice survival rate was monitored over time. The survivalrate was analyzed by Kaplan-Meier log rank test.

Example 27

FIG. 26 illustrates one embodiment of in situ one-pot fucosylation of invitro differentiated mouse dendritic cells (DCs) significantly enhancedE-selectin binding and decreased Galectin-1 binding. One-potfucosylation reaction was used to modify mouse bone marrow deriveddendritic cells according to the procedure disclosed herein. MatureBMDCs were made by culturing bone marrow progenitor cells with GM-CSFand stimulated with LPS, with high expression of CD11c and MHC II (FIGS.26A and 26B). After fucosylation, the cell surface sLacNAc wereconverted into sLeX, which could be detected through Alexa Fluor 647conjugated anti-CLA antibody (FIG. 26C). E-selectin binding was alsoincreased (FIG. 26D). By contrast, the Galectin-1 binding decreasesafter fucosylation (FIG. 26E). (A). Scheme of differentiation andmaturation of bone marrow derived dendritic cells (BMDCs). Briefly, bonemarrow cells were collected, suspended in PBS by addition of red bloodcell lysis buffer for depletion of erythrocytes, and then seeded in1×10⁶ cells/ml in the RPMI 1640 media with recombinant GM-CSF (20 ng/ml,Peprotech) in a humidified incubator with 5% CO2 at 37° C. The cellswere fed once at the interval of 3 day with the identical dose ofrecombinant GM-CSF. Immature dendritic cells were harvested on day 6with the purity of CD11c+ cells higher than 90%. Then the cells werestimulated with LPS for maturation. (B). Immature DCs were stimulatedwith LPS (100 ng/ml) for 12 h, expression of CD11c and MHC II wereanalyzed by flow cytometry. (C-E). Mature DCs were fucosylated using theone-pot strategy, then the cells were stained with anti-CLA antibody,E-selectin or Gal-1 protein, and then washed, stained with secondantibody. After the staining process, the samples were analyzed by flowcytometry. M.F.I. means “‘Medium of Fluorescence Intensity”. Data arepresented as mean ± standard deviations and analyzed for statisticalsignificance by two-tailed student’s t test in Prism (GraphPad)software. Differences with P values < 0.05 were considered statisticallysignificant. * means P < 0.05.

Example 28

FIG. 27 illustrates one embodiment of fucosylation of antigen loaded DCsinduces a significantly stronger anti-tumor immune response. To evaluatethe effect of fucosylated DCs in inducing anti-tumor immune response,B16-OVA melanoma tumor model was used (FIG. 27A). Compared withunfucosylated DC vaccine group, fucosylated DCs vaccine was found toinhibit tumor growth more efficiently. (A). Scheme of fucosylateddendritic cell vaccine inhibiting tumor growth in vivo. C57BL/6 wildtype recipient mice were inoculated subcutaneously with 5×10⁵ B16-OVAmelanoma cells in right flank, 5×10³ naive OT-I T cells were inoculatedinto the mice through retrobulbar injection on day 6 post tumor cellinoculation. On day 8, mature DCs were incubated with 200 nM OVA257-264(SIINFEKL) peptide for 1.5h, washed and then fucosylated using theone-pot strategy. After fucosylation, 4×10⁵ DCs were inoculated into thetumor bearing mice through subcutaneous injection in right flank. Thentumor growth was recorded. (B). Tumor sizes were measured by Verniercaliper and calculated. 8 mice were in each experimental group. Data arepresented as mean ± standard deviations and analyzed for statisticalsignificance Mann-Whitney non-parametric test in Prism (GraphPad)software. Differences with P values < 0.05 were considered statisticallysignificant. * means P < 0.05.

Example 29

FIG. 28 illustrates one embodiment of modification of cultured Lec2 CHOcell surface using crude one-pot GDP-fucose-DNA-FAM. One-potfucosylation reaction was used to modify CHO-lec 2 cells with apolynucleotide (a single chain DNA). GDP-fucose-DNA-FAM was synthesizedby the one-pot procedure according to the procedure in Example 1. TheDNA substrate is a single chain DNA with a 5′ alkyne group and a 3′ FAMfluorophore. The term “FAM” or “6-FAM,” as used herein, refers to thefluorescent dye 6-carboxyfluorescein. Using GDP-fucose-DNA-FAM assubstrates, the cell surface was functionalized with a single chain DNAspecifically on LacNAc, which could be confirmed by the fluorescentsignal on the DNA (FIG. 28 ). The fucosylation protocol is the same asin Example 3. As shown in FIG. 28(B), the lec2 cells fucosylated withGDP-fucose-DNA-FAM shows a significant increase in the fluorescence ofFAM. Control experiments were also characterized without 1,3 FucT (FIG.28(B)), which indicates that the signal is from the fucosylationreaction.

Example 30

FIG. 29 illustrates one embodiment of anti-human E-selectin installed oncultured Lec2 CHO cell surface can bind to human E-selectin. Anti-humanE-selectin antibody was installed on lec2 cell surface through protocolin Example 8. After that, cells labeled with anti-human E-selectin orits isotype antibody were incubated with the human E-selectin Fcprotein. The result indicates that only the cells labeled withanti-human E-selectin can bind to E-selectin protein (FIG. 29 ), whichwas detected by staining with APC-anti-Fc antibody.

Example 31

FIG. 30 illustrates one embodiment of anti-mouse PD-L1 installed oncultured Lec2 CHO cell surface can bind to mouse PD-L1. Anti- mousePD-L1 was installed on Lec2 cell surface using the protocol described inExample 8. After that, cells labeled with anti-mouse PD-L1 or itsisotype antibody were incubated with the mouse PD-L1 Fc fusion protein.The result indicates that only the cells labeled with anti-mouse PD-L1can bind to mouse PD-L1 protein (FIG. 30 ), which was detected bystaining with APC-anti-Fc antibody.

Example 32

FIG. 31 illustrates one embodiment of Anti-mouse CTLA4 installed oncultured CHO cell surface can bind to mouse CTLA4. Anti- mouse CTLA4 wasinstalled on CHO cell surface using the protocol described in Example 8.After that, cells labeled with anti-mouse CTLA4 or its isotype antibodywere incubated with the mouse CTLA4 Fc protein. The result indicatesthat only the cells labeled with anti-mouse CTLA4 can bind to mouseCTLA4 protein (FIG. 31 ), which was detected by staining withAPC-anti-Fc antibody.

Example 33

FIG. 32 illustrates one embodiment of Anti-mouse CTLA4 installed onmouse dendritic cell surface can bind to mouse CTLA4 protein. Mouse DCcells were first incubated with anti-CD16/32 to block Fc binding. Thenanti-CTLA4 antibody was used to block the CTLA4 antigen on DC cellsurface. After that, TCO-anti-CTLA4 antibody was installed on DC surfaceusing the protocol described in Example 8. Cells labeled with anti-mouseCTLA4 or its isotype antibody were then incubated with the mouse CTLA4Fc protein. The result indicates that the cells labeled with anti-mouseCTLA4 can bind more CTLA4 protein compared to the isotype conjugatedcells, which was detected by staining with APC-anti-Fc antibody.

Example 34

FIG. 33 illustrates one embodiment of Mouse IgG installed on OT-1 CD8 Tcell surface can stay on surface for more than 24 hours. Mouse IgGantibodies were conjugated with OT-1 CD8 T cells as described in Example13. Later, APC-anti mouse IgG was used to track the T cell surfacecovalently linked mouse IgG molecules. As shown in FIG. 33 , the signalfrom mouse IgG can be obviously detected after 24 hours.

Example 35

FIG. 34 illustrates one embodiment of Conjugate OT-1 CD8 T cell withmouse IgG antibodies doesn’t affect the proliferation. Mouse IgGantibodies were conjugated with OT-1 CD8 T cells as described in Example13. Then the cells were cultured in T cell culture media with 10 ng/mlIL2. The cell counts were counted in three days. The data shows theproliferation rate of modified cells are similar as the unmodifiedcells.

Example 36

FIG. 35 illustrates one embodiment of Anti-mouse PD-L1 installed on OT-1CD8 T cell surface can stay on cell surface for more than 24 hours.Anti-PD-L1 antibodies were conjugated with OT-1 CD8 T cells as describedin Example 15 (after blocking with anti-PD-L1 antibodies, the cells werenot stained with FITC-anti rat IgG in this example). Later, APC-anti ratIgG were used to track the T cell surface covalently linked anti-PD-L1antibodies. As shown in FIG. 35 , the signal from the conjugatedanti-PD-L1 can be obviously detected after 24 hours.

Example 37

FIG. 36 illustrates one embodiment of Conjugate OT-1 CD8 T cell withanti-PD-L1 antibodies doesn’t affect the proliferation. Anti-PD-L1antibodies were conjugated with OT-1 CD8 T cells as described in Example16. Then the cells were cultured in T cell culture media with 10 ng/mlIL2. The cell counts were counted in three days. The data shows theproliferation rate of modified cells are similar as the unmodifiedcells.

Example 38

FIG. 37 illustrates one embodiment of Anti-mouse PD-L1 installed on OT-1CD8 T cell surface can bind to mouse PD-L1. Anti-mouse PD-L1 or rat IgGisotype control was installed on OT-1 T cell surface through theprotocol in Example 16 (cells were blocked by anti-PD-L1 before covalentreaction with TCO- antibodies). After that, cells labeled withanti-mouse PD-L1 or its isotype antibody were incubated with the mousePD-L1 Fc protein. The result shows that the cells labeled withanti-mouse PD-L1 can bind more PD-L1 protein compared to the isotypeconjugated cells (FIG. 37 ). The PD-L1 binding in the isotype controlgroup comes from the blocking anti-PD-L1 antibodies on T cell surface.

Example 39

FIG. 38 illustrates one embodiment of Anti-mouse PD-L1 installed on OT-1CD8 T cell surface can improve T cells’ killing function on cancercells. Anti-mouse PD-L1 or rat IgG isotype control was installed on OT-1T cell surface through the protocol in Example 16. After that, OT-1 CD8T cells with or without modifications were mixed with B16-OVA-luc cells(B16 melanoma cells with overexpressed OVA antigen and luciferasemarker) and incubated for 2 days. According to the imaging andluciferase activity assay, OT-1 cells with covalently linked anti-PD-L1antibody can induce more T clusters and kill more cancer cells comparedto OT-1 CD8 T cells or isotype labeled T cells.

Example 40

In one embodiment, disclosed herein is a one-pot method for producingGDP-Fuc derivatives efficiently and cost-effectively. The absence offacile methods for the synthesis and purification of versatile donorsubstrates in preparative scales has hindered the applications ofenzymatic glycoengineering. In fact, the inventors have previously shownthat FucT enzyme from H. pylori could accept GDP-Fuc derivatives with afunctional group (e.g. fluorescein) larger than azide at the C6 positionof the fucose. However, great loss of products in purifying one-stepengineering substrates makes the two-step procedure more preferred incell surface. To expand the scope and applications of one-stepengineering enabled by FucT, all kinds of derivatives of GDP-Fuc shouldbe easily prepared in large scale. Since the approach for GDP-Fucanalogues synthesis is nearly quantitative (>90%), it seems thatpurifications of products are not required for subsequent cell surfacereactions if all the components after reactions are biocompatible. Totest this hypothesis, the inventors prepared one-pot crude products ofGDP-Fuc using biocompatible Mg²⁺. The one-pot products are directly usedwithout purification and the efficiency is the same as pure GDP-Fuc oncell surface (FIG. 44 ). To make this one-pot protocol compatible withversatile GDP-Fuc derivatives, one-pot products of GDP-Fuc alkyne(GF-Al) or GDP-Fuc azide (GF-Az) was directly modified with differentmoieties using ligand accelerated and biocompatible copper(I)-catalyzedalkyne-azide cycloaddition (CuAAC) previously developed (FIG. 39C). See,for example, Wang, W. et al, Proc. Natl. Acad. Sci. USA 2009, 106,16096; Besanceney-Webler, C. et al Angew. Chem. Int. Ed. 2011, 50, 8051,the contents of each of which are incorporated by reference in itsentirety, including the drawings. All the reactions were in great yields(>95%) after overnight and then quenched by the copper chelator(bathocuproine sulphonate, BCS) to make the one-pot products morebiocompatible for direct use on cells. Compared to the conventionaltwo-step protocol for cell surface LacNAc labeling (enzymatic transferfollowed by cell surface click chemistry), one-step enzymatic labelingusing one-pot GF-Al-biotin are more efficient and biocompatible (FIG. 45). Besides the biotin probe, a fluorescent probe Cy3, a bioorthogonalreaction handle tetrazine (Tz) and a dye (FAM) labeled single-strand DNA(ssDNA) were also conjugated with GF-A1 or GF-Az through this one-potprotocol in high efficiency (FIG. 39C and FIG. 46 ). All these one-potproducts were later efficiently transferred to cell surface via one-stepenzymatic reactions using FucT (FIG. 47 ). These results demonstratethis one-pot protocol is robust and its products are general applicablein the subsequent cell surface reaction. Additionally, the one-pot Tzderivative made from GF-Az is more efficient than the one made fromGF-A1 (FIG. 46D). It is consistent with the inventor’s previous resultthat GF-Az is a more favorable substrate of FucT.

Example 41

In one embodiment, disclosed herein is an enzymatic transfer method fortransferring whole IgG molecules to cell surface using FucT. Through theone-pot protocol, GDP-Fuc derivatives could be easily diversified. Amongthem, GF-Az-Tz is an interesting derivative with a very powerfulbioorthogonal handle (Tz), which could be further reacted withbiomacromolecules bearing strained dienophiles. This type of reaction isthe so-called inverse electron-demand Diels-Alder reaction (IEDDA) withextremely high efficiency and superior bioorthogonality. The inventorschallenged the substrates limit of FucT from whole IgG, as it is one ofthe biggest therapeutic biomolecules. The bioorthogonal handle oftrans-cyclooctene (TCO) with a PEG linker was installed onto mAbs ortheir isotype controls via standard amine-coupling procedures. Thelabeling reagent of TCO-PEG4-NHS ester is commercially available and theprotocols are well studied. After that, mAbs bearing TCO moieties weredirectly reacted with one-pot GF-Az-Tz to generate GDP-Fuc modified IgGmolecules (GF-IgG) (FIG. 40A). One-pot products of GDP-Fuc labeled ratIgG (GF-rIgG) were first employed in the enzymatic transfer using FucTon cell surface (FIG. 40B). Lec2 CHO cells with complex/hybrid N-glycansthat mainly terminate in LacNAc units were chosen as mode cells due toits well-defined glycan complement. Surprisingly, the transfer ofGF-rIgG to Lec2 surface was efficient and specific under normal ex vivofucosylation conditions: the very positive signal of rIgG on cellsurface analyzed by flow cytometer was only detected when both FucT (60mU) and GF-rIgG (0.1 mg/ml) were added (FIG. 40C). Meanwhile, thelabeling intensity is GF-rIgG concentration dependent, which reachessaturation at 0.1 mg/ml (FIG. 40D). Most notably, the reaction couldfinish in 10 minutes even on ice (FIG. 40E). The viability of rIgGlabeled cells was similar as unlabeled cells, which further confirm thebiocompatibility of reaction and one-pot substrates (FIG. 40F). Lec8 CHOcells, which do not express LacNAc, were used as a negative control toconfirm the transfer is dependent on the reaction between GDP-Fucmoieties on IgG molecules and the LacNAc unit on cell surface. Asexpected, there was no increase of fluorescence displayed by Lec8 cellsafter reaction compared to Lec2 cells (FIG. 40G). A competitionexperiment using cells blocked by natural substrates of FucT, GDP-Fuc,was also conducted, in which the subsequent GF-rIgG labeling was almostabolished (FIG. 48 ). These results further confirmed the reaction sitesof enzymatic transfer of GF-rIgG are almost the same as GDP-Fuc on cellsurface, which have been demonstrated as LacNAc units in complex/hybridN-glycans. Besides flow cytometry analysis, confocal microscopy imageswere taken to confirm most of the labeled rIgG stay on cell surface(FIG. 40H). To show the advantages of enzymatic engineering inintroducing multiple functionalities, GF-rIgG and GF-mIgG (GDP-Fucmodified mouse IgG) were mixed at 1:1 ratio in the enzymatic reaction.Dual label of two different IgG molecules on Lec2 cell surface wasclearly detected, which is hard to be achieved in genetic engineering(FIG. 40I and FIG. 49 ).

Example 42

In one embodiment, disclosed herein is an enzymatic transfer method fortransferring of α-E-Selectin to T cells which enables firm binding toinflamed sites. Cell homing to diseased tissues is the first importantissue of a successful ACT. Briefly, the interaction between the glycanligand sialyl Lewis X (sLeX) and the selectin family (e.g. E-selectin)mediates the tethering and rolling of circulating leukocytes on thevascular cell wall, which promotes subsequent extravasation andmigration of leukocytes through the endothelium into the surroundingtissue (FIG. 41A). In patients, cytokines (e.g. TNF-α) from the inflamedsites, such as tumorous, infected or injured tissues, induces theover-expression of E-selectin on endothelial cells of nearby bloodvessels, which could help to recruit immune cells for treatment.Therefore, targeting E-selectin to induce firm binding could helpadoptive transferred cells homing to diseased tissues for an enhancedtherapy. Unlike the conventional approach to increase the amount of sLeXon cell surface, in one embodiment, the inventors have proposed thatinstallation of α-E-selectin on T cell surface could directly mediatefirm binding of T cells on endothelial cells with high E-selectinexpression level, which could bypass natural processes of tethering,rolling and firm adhesion (FIG. 41A).

To demonstrate this possibility, the inventors used clinically relevantprimary human T cells (in similar culture conditions of CAR-T). Freshlyprepared human peripheral blood mononuclear cells (PBMC) were activatedthrough anti-CD3/CD28 treatment and expanded ex vivo for around twoweeks (characterization of phenotypes in FIG. 50 ). During the cultureprocess, the LacNAc levels of human T cells were tracked via enzymaticlabeling with GF-Al-biotin (FIG. 51A). Both CD4+ and CD8+ T cells have agreat increase of LacNAc level after activation, which stay at amoderate level after two weeks (CD8+ is higher than CD4+). Afterconfirming that ex vivo expanded human T cells have enough LacNAc forlabeling, FucT was used to transfer GDP-Fuc modified anti-humanE-selectin (GF-α-hE-Sel) to human T cells as well as an isotype control,GF-mIgG. Both of these two antibodies were specifically labeled to humanT cells, while the labeling efficiency have negligible variations amongthree different donors (FIGS. 41B, 51B, 51C). After that, cellsconjugated with mIgG were stained using several surface markers andcompared with unlabeled cells. The results indicate that enzymatictransfer of IgG molecules to cell surface does not affect the surfacemarkers (FIG. 50B). Whole IgG conjugated on human T cell surface stayedmore than 24 hours (~50% left after 24 hours) and had no effect on cellsproliferation (FIGS. 51D and 51E). Labeled or unlabeled human T cellswere then mixed with hE-Selectin/human Fc Chimera protein(hE-Selectin-hFc). Compared to unlabeled cells or mIgG group, cellslabeled with α-hE-Sel strongly bind to its antigen, hE-Selectin-hFcprotein, which indicates the mAbs conjugated to cell surface still havefunctions (FIG. 41C, FIG. 51F). Meanwhile, in a flow chamber assay, Tcells with different modifications were mixed with unlabeled cellsseparately and then flowed through hE-Selectin-hFc coated slides undercertain shear stress. Firm binding to E-selectin on the slide was onlyachieved when T cells were conjugated with α-hE-Sel (FIG. 41D). Tofurther confirm the results on cell surface, CFSE labeled human T cells(green) were modified with antibodies and then added to human umbilicalvein endothelial cells (HUVEC) stimulated with or without TNF-α. Afterwashing off unbound cells, T cells binding to HUVEC were quantified byplate reader via the fluorescence intensity of CFSE. The highest signalwas shown when T cells conjugated with α-hE-Sel were added to TNF-αstimulated HUVEC (FIG. 41E). A parallel imaging experiment shown in FIG.41F has similar results. Moreover, a transwell assay was performed toshow that the transendothelial migration ability of human T cells overHUVEC monolayer was not affected by the antibody labeling (FIG. 41G).These results show that engineering human T cells via installation ofα-hE-Sel lead to firm binding of T cells on TNF-α stimulated endothelialcell with unaffected transmigration ability, which indicate thisengineering could potentially promote efficient T cells homing todiseased tissues.

Example 43

In one embodiment, disclosed herein is a method for enzymaticconstruction of conjugates between NK-92MI cells and Herceptin forenhanced cancer killing. Specific killing is the key step of ACT in cellbased cancer immunotherapy. Human natural killer (NK) cells play acrucial role in innate immunity against malignant cells, yet beingdeveloped as an effective cancer killer in cancer immunotherapy. NKcells mediated specific killing of target cell involves severalmechanisms, in which antibody-dependent cell-mediated cytotoxicity(ADCC) is one of the most important. However, one challenge of NK cellbased therapy is difficult to obtain sufficient numbers of active NKcells from a patient’s blood. NK-92, a highly cytotoxic natural killer(NK) cell line established from patients with clonal NK-cell lymphoma,can be employed to generate larger numbers of cytotoxic NK cells inGMP-grade, which is a so called “off-the-shelf therapeutic” for adoptiveNK-based cancer immunotherapy. Currently, there are four phase I trialsin US, Canada, and Germany for different malignancies conducted withnormal NK-92 cells. However, specific targeting is a golden standard fora good cancer therapy. NK-92 cells do not express Fc receptors for ADCCeffects to target specific cells, which limit its wide applications. Inone embodiment, as described herein mAbs could be enzymaticallytransferred to cell surface much easier and more efficiently thangenetic approaches. Thus, in on embodiment, construction of NK-92cell-mAb conjugates would be a great technique to achieve specifictargeting in therapy.

NK-92MI cells, an IL-2 independent variant of NK-92 cell line,constantly express high level of LacNAc, which is good for FucT basedengineering. Expectedly, transfer of GDP-Fuc modified human IgG(GF-hIgG) to NK-92MI cells surface is efficient and specific undernormal ex vivo fucosylation conditions (FIG. 52A). Herceptin, also namedTrastuzumab, is a mAb for the treatment of human epidermal growth factorreceptor 2-positive (HER2+) breast cancer. In one embodiment, NK-92MIcells conjugated with Herceptin could target HER2+ cancer cells toenable the proximity enhanced cancer killing (FIG. 42A). GF-Herceptinwas successfully transferred to NK-92MI cells surface using FucT (FIG.52B). The half-life of Herceptin displayed on cell surface is about 20 h(FIG. 52C). Herceptin conjugated to NK-92MI cells still exclusively bindto HER2 antigen (FIG. 42B, FIG. 52D, FIG. 52E). In addition, NK-92MIcells conjugated with Herceptin strongly bind to HER2+ breast cancercell BT474 in a co-culture system, while unmodified NK-92MI cells weaklybind to BT474 (FIGS. 42C and 42D). Furthermore, Herceptin labeledNK-92MI cells killed BT474 cells more efficiently than unlabeled NK-92MIcells ex vivo (FIG. 42E). Neither control hIgG labeling nor freeHerceptin co-treatment could enhance the killing activity of NK-92MI onBT474, which indicates that conjugation between Herceptin and NK-92MIcell surface is required (FIG. 42E). The enhanced killing effect ofNK92-MI-Herceptin was later confirmed in other HER2+ cancer cellsincluding SKBR3 and MDA-MB-435/HER2+, but not in HER2- cancer cells likeMDA-MB-435 and MDA-MB-468 (FIG. 42F). This specificity further confirmHerceptin conjugated NK-92MI cells enhance killing through strongbinding to HER2 antigens on cancer cell surface. Interestingly, thetotal secretion of granzyme B does not increase for the enhanced killingeffect (FIG. 52E). This result tells us prolixity between NK-92MI cellsand target cells could sufficiently induce the enhanced killing activityand specificity of NK-92MI cells. High concentration of GF-Herceptin inenzymatic reactions leads to more Herceptin conjugation as well asantigen binding on NK-92MI cells surface (FIGS. 53A and 53B). Thus, thekilling efficiency of NK-92MI cells is also dependent on GF-Herceptinconcentrations in labeling reactions (FIG. 42G). Similar to other cellmediated cytotoxicity, higher effector to target cell ratios also havebetter killing results, but only in Herceptin labeled NK-92MI group,which reaches saturation at 5:1 (FIG. 42H). Another important advantageof enzymatic engineering is that people can conjugate several antibodiesonto cell surface at one time. To show the application, NK-92MI cellswere labeled with both of Herceptin and α-EGFR (FIGS. 54A and 54B),which had better killing efficiency on SKOV3 cells (HER2+EGFR+) thaneither single mAb labeling (FIG. 42I).

As developed from a patient with lymphoma, NK-92 should be irradiatedfor clinical use. Irradiation of NK-92MI cells with 6 Gy prevented itsproliferation (FIG. 55A). Killing efficiency of irradiated NK-92MI cellsis comparable with non-irradiated ones (FIG. 42J). in one embodiment,the half-life of Herceptin conjugated to irradiated NK-92MI was found tobe slightly longer than non-irradiated group (FIG. 57B). The promisingresults of enhanced ex vivo killing effect let us try to test thissystem in vivo. In one embodiment, the inventors chose an experimentallung metastasis model as a proof of concept, in which NSG mice receivedintravenous injections of MDA-MB-435/HER2+/F-luc cells (stablytransduced with firefly luciferase). After one day of tumor cellinoculation, mice were treated by i.v. injections of HBSS buffer,parental NK-92MI or NK-92MI-Herceptin cells (irradiated NK-92MI cells).Six days after tumor challenge, the lung tumor size of mice wasdetermined by longitudinal noninvasive bioluminescence imaging. Whiletreatment with parental NK-92MI cells moderately reduced lung tumorformation (~48% smaller than HBSS group), Herceptin labeled NK-92MIcells have significantly enhanced in vivo tumor killing activity (~83%smaller than HBSS group) (FIG. 42J and FIG. 56 ). Taken together,NK-92MI-Herceptin conjugates have significantly enhanced anti-tumoreffect than parental NK-92MI cells both ex vivo and in vivo, whichshowed great potential to be translated to the clinical trial.

Example 44

In one embodiment, as disclosed herein α-PD-L1 conjugated on cellsurface is capable of blocking PD-1/PD-L1 pathway and enhance T cellproliferation ex-vivo. Beyond the applications of enhancing or inducingcell-cell interactions, the present disclosure shows that mAbsconjugated to cell surface could specifically affect the signalingpathway to enhance the therapy. First of all, OT-1 CD8+ T cells werechosen as model cells since its transgenic T-cell receptor (TCR) onlyrecognize OVA₂₅₇₋₂₆₄ peptide presented by the MHC I molecule, which is agood model to study CD8+ T cell response to specific antigen. The OT-1splenocytes were activated by OVA peptides and ex vivo expanded byadding IL2 or IL7/IL15. After three days, most of the cells are CD8+ Tcells (FIG. 57A), while their LacNAc level is high enough for labelingafter activation (FIG. 57B). To exclude the possibility that large IgGmolecules on cell surface would block TCR signaling nonspecifically,OT-1+/-CD45.1+/- T cells conjugated with rIgG were restimulated usingOVA peptide-pulsed wild type B6 splenocytes (FIG. 57C). According to theresults of CFSE dilution, IgG molecules do not block the interactionbetween TCR and MHC I since labeled T cells have similar proliferationrate as unlabeled (FIG. 57D).

While T cell activation depends on TCR signaling, additionalco-stimulatory signals also fine-tune this response. For example, theinteraction between programmed death 1 (PD-1) receptor and PD-Ligand(PD-L) pathway inhibit T cell activation (FIG. 43A), which is a reasonof cancer immune evasion. Therefore, in one embodiment, installation ofα-PD-L1 on T cell surface could specifically block the PD-1/PD-L1interaction to enhance the activation of T cells and the subsequentcancer killing effect (FIG. 43B). TCR-T cells (OT-1 or P14) were labeledefficiently using FucT and GF-α-PD-L1 (FIGS. 58A and 58B). After that,the antigen binding capacity of α-PD-L1 on T cell surface was furtherconfirmed (FIG. 43C and FIG. 58C). As expected, the intensities oflabeling and antigen binding are both GF-α-PD-L1 concentrationsdependent (FIG. 43D and FIG. 58D). Then OT-1 CD8+ T cells with differentmodifications were subjected to an ex vivo killing assay, in which aB6-derived melanoma cell line B16F10 expressed ovalbumin (B16-OVA) wasused as antigen-specific cancer cells. After 20 hours of incubation,OT-1 T cells conjugated with α-PD-L1 showed a significantly enhancedcytotoxic activity on B16-OVA cells, while isotype rIgG labeling hassimilar results as unlabeled (FIG. 43E). Remarkably, anotherTCR-transgenic CD8+ T cells (P14) of irrelevant specificity were alsoconjugated with α-PD-L1 as a negative control (FIG. 43E), which showedthat α-PD-L1 itself could not induce the killing without a specificTCR-T. This synergistic killing effect indicates that α-PD-L1 conjugatedto CD8+ T cells improves the TCR specific cytotoxicity through thePD-1/PD-L1 pathway blockade. Moreover, in different effector to targetcell ratios (>1:1), the enhanced killing effect (FIG. 43F) could beseen. Unlike the antigen binding saturated at ~ 0.1 mg/ml GF-α-PD-L1(FIG. 43D), the enhanced killing effect reached the maximum at ~ 0.05mg/ml GF-α-PD-L1 (FIG. 43G), which indicates that only half of themaximum labeling could efficiently block the PD-1/PD-L1 pathway.

To further confirm these α-PD-L1/ CD8+ T cell conjugates could escapefrom the PD-1/PD-L1 coinhibitory signaling, the inventors measuredcytokine secretions of OT-1 T cells when mixed with B16-OVA cells. Theenhanced IFN-γ and TNF-α secretion were only observed in T cellsconjugated with α-PD-L1, which were also α-PD-L1 labeling intensitydependent (FIG. 43H and FIG. 59 ). The blockade of co-inhibitorysignaling promotes the T cell activation that leads to more cytokinesecretion. This kind of enhanced T cell activation also could bedirectly observed in the microscopy, as the clusters of T cells weremuch bigger than others in the α-PD-L1 labeling group (FIG. 43I and FIG.60A). Furthermore, the enhanced T cell activation also promoted T cellproliferations, which is confirmed by a CFSE dilution assay ofre-stimulating OT-1 T cells with B16-OVA (FIG. 43J and FIG. 60B). Afterconfirming the α-PD-L1 labeling could last more than 24 hours (FIG.58E), a competition assay was designed to test if α-PD-L1/ CD8+ T cellconjugates could have a better proliferation rate than unlabeled cellsin vivo. OT-1 CD8+ T cells with a congenic marker (OT-1+/-CD45.1+/-)were conjugated with α-PD-L1 and then mixed with another congenic markerlabeled OT-1 CD8+ T cells (OT-1+/-Thy1.1+/-) in a ratio around 1:1.Similarly, OT-1+/-Thy1.1+/- T cells were conjugated with α-PD-L1 andthen mixed with unmodified OT-1+/-CD45.1+/- T cells. These two groups ofOT-1 T cells were injected into B16-OVA challenged B6 mice through tailvein separately. After 48 hours of expansion, the ratio of two differentcongenic markers labeled OT-1 cells in two groups were analyzed.However, the ratios in blood, draining lymph nodes (dLN) and tumor haveno significant difference between two groups (FIG. 43K), which indicatesthe co-inhibitory signaling in tumor microenvironment are much morecomplicated than ex vivo.

Example 45

In one embodiment, as disclosed herein, enzymatic construction ofconjugates between NK-92MI cells and Herceptin enhances cancer killingby NK cells in intraperitoneal cancer model in vivo. An intraperitonealtumor model was set up by injecting MDA-MB-435/HER2+/F-luc cells throughi.p injection. On day 7 and day 10 post tumor inoculation, mice weretreated by i.p. injections of HBSS buffer, parental NK-92MI orNK-92MI-Herceptin cells (irradiated NK-92MI cells). Tumor growth wasmonitored by longitudinal noninvasive bioluminescence imaging (FIG.61A). Herceptin labeled NK-92MI cells enhanced killing of tumorsignificantly compared with NK-92MI cells treatment group (FIGS. 61B,61C and 61D). Taken together, NK-92MI-Herceptin conjugates havesignificantly enhanced anti-tumor effect than parental NK-92MI cellsboth ex vivo and in vivo, which showed great potential to be translatedto the clinical trial.

Example 46

In one embodiment, as disclosed herein, the cancer-associated antigenMUC1, a large (> 250 kDa) heavily glycosylated type 1 transmembraneprotein, is overexpressed and modified by tumor cells in over half ofall cancer cases. The 16A antibody (mouse) reacts with human MUC1. Inone embodiment, 16A-NK-92MI conjugates were constructed through theone-pot fucosylation method as disclosed herein. 16A was conjugated toNK-92MI cells only when treated with GF-16A and FT, which is also GF-16Aconcentration dependent (FIGS. 62A and 62B). Breast cancer cell lineSKBR3 has more MUC-1 antigens on cell surface than MDA-MB-435 (FIGS. 62Cand 62D). NK-92MI cells modified with 16A induced the lysis of SKBR3cells more effectively than unmodified NK-92MI cells (FIG. 62E). Neithertreated with GF-16A without FT nor co-treatment with excess free 16A (5µg/m1) could enhance the killing activity of NK-92MI on SKBR3 cells,indicating that covalent conjugation of Herceptin to the surface ofNK-92MI cells is required (FIG. 62E). By contrast, there are no enhancedkilling effects of 16A-NK-92MI conjugates on MDA-MB-435 cells since itsMUC1 expression is low. In all killing experiments, NK-92MI cells weretreated with 0.2 mg/ml GF-16A. Cancer cells were mixed with NK-92MIcells or its conjugates for 4 hours in the ratio of ½. This experimentdemonstrated the ACC strategy for enhanced killing activity of NK-92 MIcells could be extended to other antigens beyond HER2.

Example 47

In one embodiment, the enzymatic biomacromolecules transfer as disclosedherein was extended from fucosylation to sialylation (FIG. 63A). Severalsialyltransferases have been demonstrated by other groups that couldtransfer CMP-sialic acid derivatives to cell surface. However,CMP-sialic acid conjugated to proteins, like whole IgG molecules, arenot in the previous substrate scopes of these enzymes. We firstsynthesized CMP-sialic acid-azide-tetrazine (CS-Az-Tz) and CMP-sialicacid-propargyl carbamate-tetrazine (CS-Poc-Tz) using one-pot clickchemistry (FIG. 63B). Then, mouse IgG2a antibodies bearing TCO moietieswere reacted with CS-Az-Tz or CS-Poc-Tz via the inverse electron-demandDiels-Alder reaction (IEDDA) to generate CMP-Sialic acid-conjugated IgGmolecules (CS-IgG) (FIG. 63C). The one-pot product of CMP- Sialic acidconjugated mouse IgG (CS-mIgG) was then incubated with NK-92 MI cellsthat express abundant terminal LacNAc units in the presence ofsialyltransferases (STs) (FIG. 63D). We tested three STs, includingrecombinant ST6Ga11, Pasteurella multocida α(2,3) sialyltransferaseM144D mutant (Pm2,3ST-M144D), Photobacterium damsel α(2,6)sialyltransferase (Pd2,6ST). According to the results, all of thesethree enzymes could transfer CS-mIgG to cell surface (FIGS. 63E and63F). In general, CS-Az-IgG is a more favorable substrate of these STsthan CS-Poc-IgG. ST6Ga11 is the most efficient ST in these three enzymes(FIGS. 63E and 63F). Taken together, ST6Ga11 and CS-Az-IgG is the bestpair to transfer IgG to cell surface through sialylation (FIG. 63E).This example demonstrates cell surface engineering through glycan toinstall biomacromolecules could also be achieved usingsialyltranferases. All the applications in this disclosure using FucTwould be similarly achieved through ST.

Example 48

In one embodiment, the present disclosure provides a one-step enzymaticACC construction method through cell surface glycoengineering on LacNAc,which is fast and easy to use. This method is also cost effective due tothe one-pot synthesis protocol of GDP-Fuc derivatives, as disclosedherein. This method is demonstrated in at least three different immunecells including primary cells and cell line, which shows its generalapplicability. Moreover, reagents of FucT and GDP-Fuc modifiedbiomolecules (e.g. GF-IgG) used in this method are stable at 4° C. formore than one month. In one embodiment, this method would be widely usedin cell surface engineering due to these attractive properties. In oneembodiment, enzymatic engineering cell surface with whole IgG moleculesin one step is a breakthrough because it shows more possibilities ofnon-genetic cell surface engineering using biomacromolecules (e.g.enzymes, proteins, virus and even cells), especially in therapeuticapplications.

Although mAbs conjugated on cell surface would be diluted because ofproliferations and internalizations (half decay time: 8-24 h inexperiments), these transient modifications could have a lot ofapplications and serve as complements to the permanent geneticengineering approach. Other advantages, such as multiple mAbsinstallations at one time, making the instant enzymatic method moreappealing in many cases than genetic engineering. In one embodiment, theinventors have applied the new methods disclosed herein to easilyconstruct three ACCs, which have potentials to enhance ACT in threedifferent stages: homing, targeting and signaling. Among these,NK-92MI-Herceptin conjugates stand out since it has been demonstrated inkilling HER2+ cancer cells both ex vivo and in vivo. As NK-92MI cellsare in clinical trials and Herceptin is a commercial drug, theNK-92MI-Herceptin conjugates disclosed herein would be easier inclinical translation. Moreover, NK-92MI cells could be easily conjugatedwith other therapeutic mAbs via this method, which may find broadapplications in cancer therapy. In one embodiment, growing mAbs andcell-based therapies could be easily combined to get synergistic effectusing the methods disclosed herein.

Example 49

Materials and Reagents: All chemical reagents and solvents were obtainedfrom Sigma-Aldrich and used without further purification unlessotherwise noted. All cell culture materials are listed in cell culturemethods. ImmunoCult human CD3/CD28 T cell activator was purchased fromSTEMCELL Technologies, Inc. Recombinant mouse IFN-γ, recombinant mousePD-L1-human Fc Chimera (PD-L1-hFc), CFSE cell division tracker kit, APCStreptavidin, anti-mouse CD3 (17A2)-FITC, anti-mouse CD8 (53-6.7)-PE,anti-mouse CD45.1 (A20)-Pacific blue, anti-mouse Thy1.1 (OX7)-AlexaFluor 700, anti-mouse IgG (Poly4060)-APC, anti-human Fc (HP6017)-APC,anti-rat IgG (Poly4050)-APC, anti-His Tag (J095G46)-PE, anti-human CD3(HIT3a)-APC, anti-human CD45 (HI30)-FITC, anti-human CD4(RPA-T4)-PE/Cy7, anti-human CD8 (SK1)-Pacific Blue, anti-human CD25(M-A251)-PerCP/Cy5.5, anti-human CD44 (BJ18)-FITC, anti-human CD45RO(UCHL1)-Alexa Fluor 700, anti-human CD62L (DREG-56)-PE and human FcReceptor blocking solution were purchased from Biolegend. Bulkymonoclonal antibodies including anti-human E-selectin (α-hE-Sel, CL2),isotype mouse IgG2a (C1.18.4), anti-mouse PD-L1 (α-PD-L1, 10F.9G2),isotype rat IgG2b (LTF-2) and anti-human EGFR (528) were purchased fromBio X Cell. Therapeutic Herceptin were from Genentech. The control humanIgG was obtained from Athens Research and Technology. Recombinant humanE-Selectin/CD62E Fc chimera protein (hE-Selectin-hFc) and recombinanthuman TNF-α protein were purchased from R&D systems, Inc. Recombinanthuman HER2/ErbB2 Protein with His Tag (HER2-His) was purchased from SinoBiological, Inc. Click reagents including biotin-PEG3-azide (AZ-104, MW:444.5), methyltetrazine-PEG4-azide (1014, MW: 389.40),methyltetrazine-PEG4-alkyne (1013-old, MW: 487.5, a discontinued productin their website), TCO-PEG4-NHS Ester (A137, MW: 514.6) and Cy5-TCO(1089, MW: 959.20) were purchased from Click Chemistry Tools LLC.Cy3-Azide (MW: 712.8) is a gift from Prof. Xing Chen’s lab (PKU, China).(5OctdU)-5′-CAGTCAGTCAGTCAGTCAGT-3′(6-FAM) was ordered from IntegratedDNA Technologies, Inc. D-Luciferin (monosodium salt), TNF-α mouse ELISAkit, IFN-γ mouse ELISA kit, Granzyme B human ELISA kit, DiD′ solid,Hoechst 33342, DAPI, Alexa Fluor 647 NHS Ester, cell tracker green(CMFDA) and orange (CMTMR) were purchased from Thermo Fisher Scientific.CytoSelect™ Leukocyte Transmigration Assay kit and 5×RIPA buffer kitwere purchased from Cell Biolabs, Inc. CytoTox 96® non-radioactivecytotoxicity assay and Bright-Glo™ luciferase assay system werepurchased from Promega Corporation. Inorganic pyrophosphatase, FKP andFucT were expressed in endotoxin free bacteria (ClearColi® BL21-DE3,Lucigen) and purified as previously reported. Pure GDP-Fuc, GF-Al andBTTP were synthesized as previously described.

Equipments: All of the flow cytometry analyses were performed on anAttune NxT Flow Cytometer. Images of protein gels including coomassieSDS-PAGE gel and western blotting membrane were taken on ChemiDoc XRS+(Bio-Rad). Absorbance, fluorescence intensity and luminescence weremonitored in a Multi-Mode Microplate Reader (Synergy™ H4, Bio-Tek).Confocal images were taken on a Nikon spinning disk confocal microscope(TE2000). Fluorescent and phase contrast microscope images were taken onan All-In One fluorescence microscope (Keyence, BZ-X700).Bioluminescence imaging of live mice were acquired using an IVISSpectrum system.

Cells: Cell lines are all from ATCC unless otherwise specified. CHO celllines (WT, Lec2 and Lec8, from Prof. Pamela Stanley lab) were grown asmonolayer in alpha-Minimum Essential medium (α-MEM) (GIBCO) supplementedwith 10% fetal bovine serum (FBS) (Omega Scientific, Inc). HUVEC cellline was cultured in RPMI 1640 (GlutaMAX, GIBCO) with 20%heat-inactivated FBS (Omega Scientific, Inc), 40 µg/mL endothelial cellgrowth supplement (Corning), 100 µg/mL heparin (Sigma-Aldrich), 50 µMβ-ME (GIBCO), and 10 mM HEPES (GIBCO). HUVEC culture dish was pre-coatedwith 0.1% gelatin solution (ATCC) at 4° C. overnight. NK-92MI cell linewas grown in MyeloCult™ H5100 (STEMCELL Technologies, Inc). Cancer celllines including BT474, SKBR3, MDA-MB-435 (HER2+ and HER2-), MDA-MB-468,SKOV3 and mouse B16-OVA (from Prof. Gregoire Lauvau lab) are all grownin grown in DMEM (Dulbecco’s modified Eagle’s medium, GlutaMAX, GIBCO)supplemented with 10% FBS. Human blood samples were collected fromhealthy donors under the TSRI Normal Blood Donor Services program (#IRB15-6710). Peripheral blood mononuclear cells (PBMCs) were obtained byFicoll (Ficoll-Paque Plus, GE) density centrifugation. PBMC, activatedhuman T cells and mouse T cells were all cultured in RPMI 1640(GlutaMAX) with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 50 µMβ-ME, 10 mM HEPES and 1×MEM NEAA (GIBCO) (referred as T cell culturemedia later). Cytokines in T cell culture media were added as indicated(rhIL2, rhIL7 and rhIL15 are all from NIH program). All cells cultureswere incubated at 37° C. under 5% CO₂.

Mice: All mice were bred or housed under specific pathogen free (SPF)conditions. All animal experiments were approved by TSRI Animal Care andUse Committee. OT-1 mice are purchased from Taconic Biosciences. CD 45.1and Thy 1.1 mice from C57BL/6J (B6) genetic background were purchasedfrom the Jackson Laboratory. Strains of OT-1+/-/CD 45.1+/- andOT-1+/-/Thy 1.1+/- were generated by cross breeding. Six female B6background P14 mice were gifts from Prof. John Teijaro lab. 176 femaleNSG mice are gifts from Prof. Philippe A. Gallay lab. Both male andfemale mice of 8-12 weeks of age were used for most experiments.

One-pot protocol for producing GDP-Fuc derivatives: Reactions weretypically carried out in a 15 mL corning tube with 5 mL 100 mM HEPESbuffer (pH 7.5) containing L-fucose analogues (final concentration, 10mM), ATP (10 mM), GTP (10 mM), MgSO₄ (10 mM), KCl (50 mM), inorganicpyrophosphatase (90 units, ~0.17 g/L, endotoxin free), and FKP (9 units,~0.6 g/L, endotoxin free). The reaction mixture was incubated at 37° C.for 5-6 h with shaking (225 rpm). After the reaction finished (monitoredby TLC analysis), enzymes were precipitated by adding 5 mL cold EtOHinto the crude product. After the precipitates were removed throughcentrifuge (8000×g, 5 min), the crude products (containing ~10 mMGDP-Fuc analogues) could be directly used. For GF-A1 and GF-Az, furthermodification could be achieved through CuACC reaction. Crude GF-Al/GF-Azsample (5 mM, in HEPES buffer) were reacted with azide/alkyne probes (5mM) in the presence of Cu/BTTP (½, 500 µM) and sodium ascorbate (2 mM)at 30° C. for 6 h (For Tz substrates, add one volume of MeOH in reactionmixture). After reaction finished (monitored by TLC and LC-MS analysis),BCS (bathocuproine sulphonate, 2 mM) were added to quench the reaction.Following this protocol, one-pot products of GF-Al-Biotin, GF-Al-Cy3,GF-Al-Tz and GF-Az-Tz were made. Their structures are shown in FIG. 45 .

General procedure for enzymatic transfer of GDP-Fuc derivatives to cellsurface: Live cells (10~20 million) were resuspended in 100 µL HBSSbuffer containing 20 mM MgSO₄, 3 mM HEPES, 0.5% FBS, 100 µM GDP-Fucderivatives and 30 mU FucT (~0.02 mg/mL). After the incubation for 20minutes (works from on ice to 37° C.), the cells were washed with PBSand ready for further application or analysis. For biotin detection, thecells were stained with APC Streptavidin after reaction. For Tzdetection, the cells were reacted with 20 µM TCO-Cy5 on ice for 30minutes and washed three times.

Preparation of GDP-Fuc modified antibodies: All of the antibodies(full-length IgG, MW: ~150 KDa) for conjugation were first desalted intoPBS and concentrated to a 6 mg/mL solution. TCO group was firstintroduced onto antibodies according to the standard labeling protocolof TCO-PEG4-NHS ester and a previous report about its application on IgGlabeling. Briefly, fresh 10 mM stock of TCO-PEG4-NHS reagent in DMSO wasprepared, and added it to the IgG sample (final concentration 6 mg/mL)at a final concentration of 0.5 mM. The reactions were incubated at roomtemperature for 30 minutes and quenched by adding Tris buffer (pH 8.0)to a final concentration of 50 mM Tris. The quenched reaction mixtureswere incubated at room temperature for 5 minutes and then desalted intoPBS using desalting column (GE). The concentrations of desalted TCO-IgGswere around 4.5 mg/mL (~4 TCO per IgG). After that, ~10 mM one-potproducts of GF-Az-Tz were added to TCO-IgGs with a final concentrationat 0.15 mM (5eq of IgG). After 30 minutes of incubation at roomtemperature, these one-pot GF-IgG products were ready to use and couldbe kept at 4° C. for up to 2 months. For Alexa Fluor 647 labeled GF-rIgGused in confocal imaging, the rIgG molecule was first labeled with AlexaFluor 647 probes following the manual and then subjected to thisprotocol. According to the reference, these mAbs modified by NHS esterand subsequent IEDDA reaction have similar antigen binding affinity asunmodified mAbs.

General procedure for enzymatic transfer of GF-IgG to cell surface: Livecells (10~20 million) were resuspended in 100 µL HBSS buffer containing20 mM MgSO₄, 3 mM HEPES, 0.5% FBS, 0.1 mg/mL one-pot GF-IgG (differentconcentrations were used in titration) and 60 mU FucT (~0.04 mg/mL).After the incubation for 20 minutes (works from on ice to 37° C.), thecells were washed with PBS and ready for further application oranalysis. For labeling detection, IgG labeled cells were stained withDAPI and fluorescent secondary antibody against labeled IgG (1/50-1/200dilution) on ice for 30 minutes. For confocal imaging, Lec2 cells platedon a chamber cover glass (Nunc) were treated with 0.1 mg/mL Alexa Fluor647 labeled GF-rIgG in the same fucosylation condition as describedabove. After conjugation, live cells were washed and stained withHoechst 33342 for 30 minutes on ice and then washed for imaging. Toconfirm the binding activity of cell surface conjugated antibodies,labeled cells were allowed to bind with 10 µg/mL antigen (hE-sel-hFc,PD-L1-hFc or HER2-His) in binding buffer (HBSS with 5 mM HEPES, 2 mMCaC1₂, and 1 mM MgC1₂) on ice for 30 minutes. After binding, cells werewashed twice with PBS and stained with DAPI and APC-anti-human Fc (1/50dilution) or PE-anti His (1/100 dilution).

Primary human T cells preparation and IgG labeling on their surface:Fresh PBMCs were freshly prepared as described above. 4 million per mLPBMCs were cultured in T cell culture media with 15 ng/mL rhIL2 andactivated with human CD3/CD28 T cell activator for two days. After that,activated human T cells were kept under 4^(∗)10⁶ cells/mL in T cellculture media (fresh media with cytokine were added every two days).Phenotypes were characterized after two weeks expansion (>95% are humanT cells). LacNAc levels on CD4+ and CD8+ T cells were tracked throughfucosylation with GF-biotin on day 0 (naïve T cells), day 2, day 4, day7, day 11 and day 13. Activated human T cells were labeled with α-hE-Seland mIgG control using the general procedure for enzymatic GF-IgGtransfer. After that, the labeling detection and the antigen(hE-sel-hFc) binding were both confirmed. Labeled human T cells werethen cultured in T cell culture media at a start cell density of0.5^(∗)10⁶ cells/mL. The decay of cell surface mIgG molecule was trackedin 24 hours after labeling (anti-mouse IgG staining). The cellproliferation rate of labeled T cells was compared with unlabeled humanT cells in three days (live cell counting).

Flow chamber assay on E-selectin coating slides: Sterile cover glassslides (catalog# 12-545 M, Fisher Scientific) were coated withrecombinant human E-selectin-Fc (2 µg/mL) for 2 h at room temperature,followed by blocking with casein for at least 30 min. Ibidisticky-Slides I 0.1 Luer (H: 150 µm, W: 5 mm, ibidi) were mounted on thecoated glass slides and connected with inlet and outlet tubing. Twogroups of human T cells were mixed (unmodified human T cells stainedwith cell tracker green and mIgG or α-hE-Sel conjugated human T cellsstained with cell tracker orange) and then perfused through the inlettube at a concentration of 2×10⁶ cells/mL. The outlet tube was connectedto a syringe pump (Harvard Apparatus). Wall shear stress was determinedas described before (Abadier et al., 2015, Coisne et al., 2013). Firstthe cells were allowed to settle down for 1 min at 1 dyn/cm² foraccumulation in the field of view (FOV) and started imaging acquisition.The shear stress was then enhanced to 10 dyn/cm² for 3 min. To study thebehavior of T cells, image acquisition was performed at 10xmagnification with an inverted Zeiss Axiovert 200 M (Zeiss, Feldbach,Switzerland). The numbers of firm binding cells (unmoved during the 2min acquisition after shear increase at 10 dyn/cm²) was quantified byImageJ software (National Institute of Health, Bethesda, MD).

Analysis of human T cells binding on HUVEC surface: 2,000 HUVEC wereseeded in each well of a gelatin-coated 96-well plate and grown toconfluence. HUVEC were either untreated or stimulated with 200 U/mLTNF-α for 4 h. Human T cells stained with CFSE were labeled with mIgG orα-hE-Sel using general GF-IgG transfer protocol. Labeled cells werewashed with PBS and resuspended to 10⁶ cells/mL in serum free RPMI 1640.After media in HUVEC was removed, 100 µl CFSE stained human T cells (10⁶cells/mL) of three groups (unlabeled, mIgG and α-hE-Sel) were added tountreated or treated with TNF-α. The cells were allowed to attach for 20minutes at 37° C. Unbound human T cells were removed and each well wasgently washed with warm RPMI twice. Attached CFSE stained human T cellswere lysed using RIPA buffer. The attached T cell numbers in each wellwere quantified through the CFSE fluorescence signal (ex 492 nm/ em 517nm) using a plate reader. For imaging analysis, HUVEC were stained priorto T cells binding (5 µg/mL DiD and 2 µg/mL Hoechst 33342 in culturemedia, 37° C. for 20 minutes). HUVEC were washed with warm PBS threetimes before adding CFSE stained T cells. After binding, attached cellswere replenished in live cell imaging solution and imaged with afluorescence microscope.

Transmigration assay of human T cells: Following the provided protocol,Leukocyte Transmigration Assay Kit was employed to perform transwellassay. In brief, 50,000 HUVEC cells in 300 µL HUVEC growth medium wereadded to the insert of a 24-well plate with 500 µL growth medium inbottom. Cells were grown to confluence and treated with 200 U/mL TNF-αfor 4 h. 10⁶/mL human T cells were stained with LeukoTracker in serumfree medium (RPMI with 0.5% BSA, 2 mM CaCl₂, and 2 mM MgCl₂) for 1 h at37° C. and washed three times with serum free medium. Stained T cellswere labeled with mIgG or α-hE-Sel and resuspended at 10⁶ cells/mL inserum free medium. Transwell inserts were moved to new wells containing500 µL of fresh T cell medium in the bottom, and medium in the insertwas replaced with 300 µL of T cells suspension. After incubation at 37°C. for 4 hours, migrated T cells in the bottom were lysed and theresulting fluorescence signal was measured using a plate reader (ex 480nm/em 520 nm).

IgG labeling on NK-92MI cells: NK-92MI cells or irradiated NK-92MI cells(6 Gy) were labeled with Herceptin or control human IgG according to thegeneral protocol. After labeling, the labeling detection and the antigen(HER2-His) binding were both confirmed. NK-92MI cells with or withoutIgG conjugation were then cultured in T cell media with a start celldensity of 0.5^(∗)10⁶ cells/mL. The decay of ell surface conjugatedHerceptin were tracked at day 0, day 1 and day 2 (anti-human Fcstaining). The proliferation rates of cells were tracked at day 0, day1, day 2 and day 3 (DAPI staining and FACS counting). For dualantibodies labeling, NK-92MI cells were conjugated with GF-α-EGFR firstand then conjugated with GF-Herceptin after washing.

Flow and imaging analysis of binding between NK-92MI and BT474: For flowcytometry analysis, NK-92MI cells were stained with CFSE and thenlabeled with Herceptin or not according to the general protocol. BT474cells were stained with DiD first and then mixed with Herceptin labeledor unlabeled NK-92MI cells at the ratio of 1:1. Two hours later, thecells mixture was analyzed by flow cytometry. For fluorescence imaging,BT474 cells were stained with CFSE and cultured in glass bottom petridish overnight. NK-92MI cells were then stained with cell tracker orangeand then labeled with Herceptin or not. NK-92MI cells were added toBT474 culture at ratio of 1:1. Two hours later, the co-cultured cellswere imaged by fluorescent microscope before and after PBS wash.

Analysis of NK-92MI cells-mediated cytotoxicity against HER2+ cancercells: Labeled or unlabeled NK-92MI cells were co-cultured withdifferent type of cancer cells at indicated effector/target ratios for 4hours in a 96-well plate. In most of the experiments, theeffector/target ratio is 5/1. Free Herceptin were added at a finalconcentration of 5 µg/mL if indicated. Specific cancer cell lysis wasdetected by LDH secretion in supernatant (CytoTox 96, Promega). Set-upof control groups and calculations of specific lysis were according tomanufactory’s instruction. Supernatant of each group were also collectedand subjected to granzyme B ELISA kit for quantification.

Mice model of NK-92MI mediated killing of HER2+ cancer: Thirty femaleNSG mice (6-8 weeks old) were inoculated with 5^(∗)10⁵ MDA-MB-435HER2+/F-luc cells through tail vein injection. One day later, mice wererandomly divided into three groups (10 mice per group). Each group weretreated with HBSS, NK-92MI or Herceptin labeled NK-92MI cells throughtail vein injection (3^(∗)10⁶ NK cells each mice). Six days after tumorchallenge, mice were injected with 200 µL D-luciferin (15 mg/mL) throughi.p. injection. Twelve minutes later, the bioluminescence signal in micewere analyzed by PerkinElmer IVIS system. The total photons indicatingthe tumor mice were quantified by IVIS software.

Primary OT-1 CD8+ T cells preparation and IgG labeling on their surface:Splenocytes from OT-1 mice were activated by 1 nM OVA peptides in T cellmedia for two days. After that, activated cells were in vitro expandedin fresh T cell media with 15 ng/mL rhIL2 or 10 ng/mL rhIL7 and 20 ng/mLrhIL15 for several days (kept under 8^(∗)10⁶ cells/mL, fresh media withcytokine were added every two days). Phenotypes were characterized (>95%are OT-1 CD8+ T cells). LacNAc levels on OT-1 T cells (cultured with twodifferent cytokines) were tracked through fucosylation with GF-biotin onday 0 (naïve T cells), day 2, day 4, day 7, day 9, day 11 and day 13.Activated OT-1 T cells were labeled with α-PD-L1 and mIgG control usingthe general procedure for enzymatic GF-IgG transfer. After that, thelabeling detection and the antigen (PD-L1-hFc) binding were bothconfirmed. Labeled OT-1 T cells were then cultured in T cell culturemedia at a start cell density of 0.5^(∗)10⁶ cells/mL. The decay of cellsurface α-PD-L1 molecule was tracked in 24 hours after labeling(anti-rat IgG staining).

OT-1 T cells re-stimulation using OVA₋pulsed splenocytes: Splenocytesfrom B6 WT mice were pulsed with SIINFEKL (OVA) peptide (1 µg/mL, in Tcell media) for 2 h. After that, cells were washed three times beforeuse. OT-1 T cells with CD45.1 congenic marker were stained with CFSE andthen labeled with rIgG. 10⁵ OT-1 T cells (labeled or unlabeled) weremixed with 10⁶ OVA-pulsed splenocytes in 500 µL T cell media. Controlgroups were also set up without splenocytes. The cell mixtures werecultured for 3 days. After that, cells were stained with APC anti-CD45.1and DAPI. The CFSE dilution signals were analyzed on live CD45.1positive cells.

Analysis of OT-1 CD8+ T cells mediated cytotoxicity against B16-OVA:B16-OVA cells (stably transduced with firefly luciferase) were seeded in96-well plate and treated with 10 ng/mL IFN-γ overnight (B16-OVA cellswere all treated with IFN-γ to induce high expression level of PD-1 inthis work). Labeled or unlabeled OT-1 cells were co-cultured withB16-OVA cancer cells at indicated effector/target ratios for 20 hours ina 96-well plate. In most of the experiments, the effector/target ratiois 5/1. Phenotype of T cell clusters was imaged before cell numberquantification. B16-OVA cell numbers were quantified through theluciferase activity according to the reference. The detection reagentwas directly added to medium in each well according to the manufactory’smanual (Bright-Glo, Promega). For cytokine secretion quantification,cells were cultured for 9 hours and supernatant were collected andsubjected to TNF-α and IFN-γ ELISA kit. For OT-1 T cells proliferationin killing B16-OVA, OT-1 cells were stained by CFSE before IgG labeling.Modified cells were mixed with B16-OVA cells in the effector/targetratio of 2/1. After 72 hours, the cell mixtures were stained withAPC-anti CD8 and DAPI. CFSE dilution signal was analyzed on CD8+ cells.

Competition assay for analyzing OT-1 T cells in vivo proliferationagainst B16 tumor: Splenocytes from OT-1+/-CD45.1+/- and OT-1+/-Thy.1+/-mice were activated by 1 nM OVA peptides and in vitro expanded by adding10 ng/mL IL7 and 20 ng/mL IL15 for 4 days. OT-1 T cells with differentcongenic markers were treated with or without GF-α-PD-L1 and FT. CD45.1+OT-1 T cells labeled with α-PD-L1 were mixed with Thy1.1+ OT-1 T cellsat ratio of 1:1. Similarly, unlabeled CD45.1+ OT-1 T cells were mixedwith Thy1.1+ OT-1 T cells labeled with α-PD-L1 at ratio of 1:1. Theratio of two different populations as well as successful labeling ofα-PD-L1 was analyzed by flow cytometry. The mixed T cells (10^(∗)10⁶)were injected through tail vein into B16-OVA melanoma tumor (tumor size:~ 5 mm* 5 mm) bearing mice, which were inoculated with 10⁶ B16-OVA cellsthrough subcutaneous injection on the right flank 10 days ago. After 48hours of T cells injection, blood, draining lymph node (dLN) and tumortissue of recipient mice were collected. Single cells from each tissuewere acquired, stained with anti-CD3, anti-CD8, anti-Thy1.1, anti-CD45.1fluorescent antibody and analyzed by flow cytometry. The ratio ofThy1.1+/- OT1 T cell and CD45.1+/- OT-1 T cells in these tissues werecalculated.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps, some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are the selection of constituentmodules for the inventive compositions, and the diseases and otherclinical conditions that may be diagnosed, prognosed or treatedtherewith. Various embodiments of the invention can specifically includeor exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a,” “an,” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

What is claimed is:
 1. An engineered cell, comprising an antibodycovalently bound to a N-acetyllactosamine (LacNAc) glycan on the surfaceof a cell via a sialic acid linkage.
 2. The engineered cell of claim 1,wherein formation of the sialic linkage between the antibody and thecell surface glycan is catalyzed by a sialyltransferase.
 3. Theengineered cell of claim 1, wherein the cell is a primary human T cell,a natural killer (NK) cell, a CD4+ cell, or a primary CD8+ OT-1 T cells.4. The engineered cell of claim 3, wherein the NK cell is NK-92MI cell.5. The engineered cell of claim 1, wherein the antibody is a singlechain variable fragment (scFv), fragment antigen binding (Fab) fragment,or a full length antibody.
 6. The engineered cell of claim 1, whereinthe antibody is an immunoglobulin G (IgG) antibody.
 7. The engineeredcell of claim 1, wherein the antibody is Trastuzumab.
 8. The engineeredcell of claim 1, wherein the cell is a chimeric antigen receptor (CAR)-Tcell.
 9. The engineered cell of claim 8, comprising a geneticallymodified T-cell with the cell surface LacNAc covalently bound to asialic acid bearing a new motif.
 10. An engineered cell, comprising: achemical or biological moiety covalently bound to a cell surface glycanpresent on the surface of the cell, wherein the chemical or biologicalmoiety is selected from the group consisting of small molecule,polynucleotide, polypeptide, and antibody.
 11. The engineered cell ofclaim 10, wherein the engineered cell is an immune cell.
 12. Theengineered cell of claim 11, wherein the immune cell is a T-cell or anatural killer (NK) cell.
 13. The engineered cell of claim 12, whereinthe T cell is a CD8+ or a CD4+ T cell.
 14. The engineered cell of claim11, wherein the immune cell is a Dendritic Cell (DC).
 15. The engineeredcell of claim 10, wherein the small molecule is a small drug molecule ora pharmaceutically acceptable salt or a co-crystal thereof.
 16. Theengineered cell of claim 10, wherein the chemical or biological moietyis a biotin probe, a fluorescent probe, a biorthogonal reaction handle,and/or a dye labeled single strand DNA.
 17. A method of making anengineered cell, comprising: generating CMP-sialic acid conjugated withan antibody (CS-IgG) by reacting an antibody bearing a TCO moiety withCS-Az-Tz or CS-Poc-Tz; and making the engineered cell by incubating anaturally occurring cell with a composition comprising (a) CS-IgG and(b) a sialyltransferase.
 18. The method of claim 17, wherein thesialyltransferase is selected from the group consisting of ST6Gal1,Pasteurella multocida α(2,3) sialyltransferase M144D mutant (Pm2,3ST-M144D), and Photobacterium damsel a(2,6) sialyltransferase (Pd2,6ST).19. A method of treating or inhibiting the growth of a cancer in asubject, comprising administering to the subject a therapeuticallyeffective dosage of the engineered cell, wherein the engineered cell ofclaim 1, thereby treating or inhibiting the growth of the cancer in thesubject.
 20. The method of claim 19, wherein the cell is NK-92MI, andthe antibody is Trastuzumab.